STUDIES ON CELLULAR FUNCTIONS AND MECHANISMS FOR AN ANTI-ADHESIVE SIGNAL OF PROTEOGLYCAN,PG-M

我们认为，Annexin VI作为蛋白多糖Pg-M的硫酸软骨素链的细胞表面受体，以钙依赖的方式将其抗黏附信号转导到细胞内。本文从以下两个方面对其可能性和机理进行了探讨：1)我们已成功地合成了可替代PG-M的硫酸软骨素-磷脂酰乙醇胺类脂(CS-PE)，并将其应用于包覆在盘中的防粘附基上。通过细胞黏附实验和细胞铺展实验研究了它们的抗黏附性能，旨在分别分析细胞与底物分子之间的相互作用以及随后导致细胞铺展的细胞骨架变化。就检查而言，任何不同类型的细胞都可以附着在涂有纤维连接蛋白的培养皿上，但不会在…上扩散。更多的母鸡双层涂布CS-PE。此外，细胞也可以附着在单独涂布CS-PE的培养皿上。然而，我们之前发现缺乏膜联蛋白VI的人上皮细胞株A431几乎不能附着在CS-PE涂层的培养皿上。包括A431在内的任何细胞都可以附着在涂有CS-PE以外的糖胺多糖脂的培养皿上，并随后在其上扩散。综上所述，这些结果表明，Annexin VI参与了一种特定的信号转导，以抑制随后的细胞扩散反应。不同的试剂，如蛋白激酶抑制剂，它的激活剂，和异构体对抗黏附活性有任何影响，但没有一个给出显著的影响。2)将含有不同剪接形式PG-M的DNA载体导入细胞后，分析细胞形态和迁移活性的变化，以获得具有不同潜在结合部位的硫酸软骨素链分子。转基因小鼠系统也被用来检验这些影响。然而，我们没有观察到任何显著的差异。现在重新考虑表达系统，以产生足够的表达。较少

We have suggested that annexin VI functions as a cell-surface receptor for the chondroitin sulfate chains of proteoglycan, PG-M in a Ca^<2+>-dependent manner to transduce their anti-adhesive signals into the cells. Here we have investigated the possibility and mechanisms from the following two aspects : 1) We previously succeeded in synthesizing chondroitin sulfate-phosphatidyl ethanolamine lipid (CS-PE) which can be substituted for PG-M in regard to the anti-adhesive activity and have taken advantage of dishes coated with this new molecules for anti-adhesive substrata. Their anti-adhesive properties have been investigated using both the cell attachment assay and the cell spreading assay, which are aimed preferentially to analyze the interactions between cells and substrate molecules and the subsequent cytoskeletal changes to result in the cell spreading, respectively. As far as examined, cells of any different types could attach to dishes coated with fibronectin but not spread on it w … More hen doubly coated with CS-PE.In addition, cells could also attach to dishes coated with CS-PE alone. However, A431, a cell line derived from human epithelium which we previously found to lack annexin VI could hardly attach to the CS-PE-coated dish. Any cells including A431 could attach to and subsequently spread on dishes coated with glycosaminoglycan-lipids other than CS-PE.Taken together, the results suggest the involvement of annexin VI in a specific signal transduction to inhibit the subsequent cell-spreading reaction. Various reagents such as protein kinase-inhibitors, its activators, and inophors were tested to have any effects on the anti-adhesive activity and none of them gave the significant effects. 2) Changes in cellular morphology and migration activity were analyzed when cells were transfected with the vectors containing cDNAs for various alternative spliced forms of PG-M to yield the molecules having various potential attachment sites for chondroitin sulfate chains. Transgenic mouse systems were also used to examine the effects. However, we have not observed any significant differences. The expression systems are now taken into reconsideration to yield the enough expression. Less