A study for role of PGE2 on adhesion of mast cells to fibronectin

本项目的目的是确定肥大细胞与纤维连接蛋白粘附的机制。将肥大细胞瘤P-815细胞培养于含Fisher培养基的5%- fcs预包被或未包被纤维连接蛋白的平板上，在5% c02培养箱中37℃下加PGE2孵育不同次数。得到的结果如下：1。P-815细胞响应10^<-8> M-10^<-6> M PGE2粘附在纤维连接蛋白上。PGE2的粘附活性是通过与P-815细胞上表达的EP4受体结合，随后刺激cAMP形成和cAMP依赖性蛋白激酶a活性。这些反应在PGE2加入后4-5小时内没有表现出来，这表明新形成的未知蛋白参与其中，这些蛋白随后一直在寻找。pge2诱导的P-815细胞开始粘附纤维连接蛋白后，在PKA抑制剂H-89的抑制下，粘附的P-815细胞在H89处理下不渗漏。然而，通过EGTA、BAPTA/AM和SKF96365（一种Ca2+通道ROCC抑制剂）处理，pge2粘附的P-815细胞很容易分离，细胞内Ca2+水平增加。实际上，粘附的P-815细胞被发现含有高的细胞内Ca2+水平。3 . pge2活化的P-815细胞与纤维连接蛋白的粘附在CaM激酶抑制剂的作用下受到抑制。对CAMP/ pka无关的信号进行了广泛的搜索，结果确定Epac信号（CAMP直接激活的交换蛋白）作为重要的候选分子参与。根据这些结果，我们发现pge2激活的P-815细胞粘附可能由两个信号组成，首先是在结合起始阶段cAMP/PKA依赖反应参与，而在结合维持后期Epac反应参与。这些结果对于解决肥大细胞对局部炎症组织中粘连蛋白等细胞外基质的粘附能力具有重要意义。

The purpose of this project is to determine the mechanism of mast cell adhesion to fibronectin. Mastocytoma P-815 cells were cultured on a plate pre-coated with or without fibronectin in 5%-FCS containing Fisher medium, and incubated with PGE2 for various times at 37℃ in 5% C02-incubator. Obtained results are shown below ;1.P-815 cells were adhered to fibronectin in response to 10^<-8> M-10^<-6> M PGE2. The adherence activity of PGE2 was appeared through its binding to EP4 receptors expressed on P-815 cells, followed by a stimulated cAMP formation and cAMP-dependent protein kinase A activity. These responses were not demonstrated up to 4-5 hrs after PGE2 addition, indicating involvement of newly formed unknown proteins, which have been searching thereafter.2.After initiation of PGE2-induced P-815 cell adhesion to fibronectin, being inhibited by PKA inhibitor H-89, adhered P-815 cells did not leak out by H89 treatment. However, PGE2-adhered P-815 cells were easily detached by a decrease of increasing intracellular Ca2+ level by treatment with EGTA,BAPTA/AM, and SKF96365, a Ca2+ channel ROCC inhibitor. Really, adhered P-815 cells were found to consist of high intracellular Ca2+ level.3.PGE2-activated P-815 cell adhesion to fibronectin were mostly suppressed by incubation with CaM kinase inhibitor.4.CAMP/PKA-independent signals were extensively searched for, and as a result have determined involvement of Epac signal (exchange protein directly activated by cAMP) as important candidatet molecules.From these results, we have presented that PGE2-activated P-815 cell adhesion might be comprised of two signals, first during binding initiation phase cAMP/PKA dependent reactions involved in while during late binding maintenance phase Epac reactions did. These results are surely important to resolve mast cell adhesion ability to extra-cellular matrix like ibronectin in local inflammatory tissues.