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

我们认为膜联蛋白VI作为蛋白多糖PG-M的硫酸软骨素链的细胞表面受体，以Ca^2+依赖的方式将其抗粘附信号传递到细胞中。本文从以下两个方面探讨了其可能性和机制：1）我们以前成功合成了硫酸软骨素-磷脂酰乙醇胺脂质（CS-PE），它可以替代PG-M的抗粘附活性，并利用这种新分子包被的盘作为抗粘附基质。它们的抗粘附特性已经使用细胞粘附测定和细胞铺展测定进行了研究，这两种测定的目的分别是优先分析细胞和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。就目前的研究而言，任何不同类型的细胞都可以附着在涂有纤连蛋白的培养皿上，但不能在其上扩散。 ...更多信息 此外，细胞也能贴附在单独包被CS-PE的培养皿上。然而，A431，一种我们以前发现缺乏膜联蛋白VI的人上皮细胞系，几乎不能附着在CS-PE包被的培养皿上。包括A431在内的任何细胞都可以附着并随后在涂有糖胺聚糖脂质而不是CS-PE的培养皿上铺展。总之，结果表明膜联蛋白VI参与了特异性信号转导，以抑制随后的细胞铺展反应。各种试剂，如蛋白激酶抑制剂，其激活剂，和inophors进行了测试，有任何影响的抗粘附活性，他们都没有给出显着的效果。2)当用含有PG-M的各种可变剪接形式的cDNA的载体转染细胞以产生具有硫酸软骨素链的各种潜在附着位点的分子时，分析细胞形态和迁移活性的变化。转基因小鼠系统也被用来检查的影响。然而，我们没有观察到任何显著差异。表达系统现在被重新考虑以产生足够的表达。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