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

抗粘基质分子的信号转导和细胞功能研究。

基本信息

批准号：
10480161
负责人：
KIMATA Koji
金额：
$ 6.91万
依托单位：
INSTITUTE FOR MOLECULAR SCIENCE OF MEDICINE, AICHI MEDICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

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.

几种具有抗粘附活性的细胞外基质分子已被报道，但其机制尚不清楚。蛋白聚糖PG-M/多功能蛋白聚糖通过其硫酸软骨素链以Ca 2+ 依赖性方式显示出这样的活性。我们首先鉴定了膜联蛋白VI作为细胞表面受体，可能参与抗粘附活性的信号转导。 FACS 分析显示，将膜联蛋白 VI cDNA 转染至 A431 细胞中，导致一些表达的膜联蛋白 VI 定位于细胞表面，并且细胞对硫酸软骨素包被的培养皿具有附着活性，而原始细胞从未显示出这种活性。此外，获得的细胞附着活性对硫酸软骨素具有特异性，但对其他糖胺聚糖不具有特异性，并且该活性需要膜联蛋白 VI 的独特结构。然而，新开发的用于阐明聚类信号分子的磁珠测定法并未检测到与细胞表面膜联蛋白 VI 相关的分子。奇怪的是，细胞质膜联蛋白 VI 与纤连蛋白包被的磁珠之间存在关联。此外，使用已知对信号转导具有抑制活性的各种试剂（例如 HA 1004），未观察到对 PG-M/versican 的抗粘附活性的显着抑制。此外，一系列证据表明新发现的 Pyk2 家族不参与其中获得CAK分子。目前，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/多功能蛋白聚糖突变体或不含硫酸软骨素的剪接变体的实验来评估原位抗粘连效果。