STUDIES ON SIGNAL TRANSDUCTION AND CELLULAR FUNCTION OF ANTI-ADHESIVE MATRIX MOLECULES.

已经报道了几种具有抗黏附活性的细胞外基质分子，但其机制尚不清楚。Pg-M/Verscan蛋白多糖通过其硫酸软骨素链以钙依赖的方式显示这种活性。我们首次确定Annexin VI是一种细胞表面受体，可能参与抗黏附活性的信号转导。将Annexin VI基因导入A431细胞，流式细胞仪分析显示部分表达的Annexin VI蛋白定位于细胞表面，细胞对硫酸软骨素包被的培养皿具有黏附活性，这是原始细胞所没有表现出的。此外，获得的细胞黏附活性是对硫酸软骨素特异的，而不是对其他糖胺聚糖特异的，而且这种活性需要膜联蛋白VI的独特结构。然而，最近发展起来的用于阐明聚集信号分子的磁珠分析显示，没有检测到与细胞表面膜联蛋白VI相关的分子。奇怪的是，细胞质中的膜联蛋白VI与纤维连接蛋白包裹的微珠存在关联。此外，已知对信号转导有抑制活性的各种试剂，如HA 1004，对PG-M/Verscan的抗黏附活性没有明显的抑制作用。此外，还获得了一系列新发现的PYK2家族分子CAK不参与的证据。为了达到这一目的，我们正在采取不同的方法，通过在细胞、组织或动物中表达PG-M/versican的突变体或不含硫酸软骨素的剪接突变体的实验，来原位评估其抗黏附效果。

Several extracellular matrix molecules with anti-adhesive activity have been reported but the mechanisms are yet unknown. Proteoglycan, PG-M/versican shows such an activity in Ca^<2+>-dependent manner through its chondroitin sulfate chain. We have firstly identified annexin VI as a cell surface receptor possibly involved in the signal transduction of the anti-adhesive activity. Transfection of annexin VI cDNA into A431 cells resulted in the localization of some of the expressed annexin VI on the cell surfaces shown by the FACS analysis and the cell attachment activity to chondroitin sulfate-coated dishes which the original cells never showed. Further, the gained cell attachment activity was specific to the chondroitin sulfate but not to other glycosaminoglycans and the unique structure of annexin VI was required for the activity. However, the newly developed magnetic bead assay for the elucidation of clustering signaling molecules revealed no detection of the molecules associating with the cell surface annexin VI.Curiously, there was the association of cytoplasmic annexin VI with fibronectin-coated beads. In addition, no significant inhibition to the anti-adhesive activity of PG-M/versican was observed with various reagents known to have inhibitory activities to signal transduction such as HA 1004. Furthermore, a line of evidences for no involvement of newly found Pyk2 family molecule, CAK were obtained. We are now taking different ways for the purpose by performing the experiments where the mutants of PG-M/versican or the spliced variants without chondroitin sulfate are being expressed in cells, tissues or animals to evaluate the anti-adhesive effects in situ.