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

本课题旨在探讨肥大细胞与纤维连接蛋白的粘附机制。将肥大细胞瘤P-815细胞在预先包被有或没有纤连蛋白的平板上在含有5%FCS的Fisher培养基中培养，并在37℃下在5%CO2培养箱中与PGE 2孵育不同时间。获得的结果如下所示;1.P-815细胞响应于10 μ <-8>M-10 μ <-6>M PGE 2而粘附于纤连蛋白。PGE 2的粘附活性是通过与P-815细胞上表达的EP 4受体结合，然后刺激cAMP的形成和cAMP依赖性蛋白激酶A活性而表现的。PGE_2诱导的P-815细胞与纤连蛋白的粘附被PKA抑制剂H-89抑制后，粘附的P-815细胞不被H89处理而漏出。EGTA、BAPTA/AM和SKF 96365（一种钙通道ROCC抑制剂）处理后，PGE 2粘附的P-815细胞容易因胞内钙水平升高而脱落。PGE 2激活的P-815细胞与纤维连接蛋白的粘附在很大程度上被CaM激酶抑制。4.广泛地寻找CAMP/PKA非依赖性信号，结果确定Epac信号参与其中这些结果表明，PGE_2激活的P-815细胞粘附可能由两个信号组成，首先在结合起始期参与cAMP/PKA依赖性反应，而在结合维持后期参与Epac反应。这些结果对了解局部炎症组织中肥大细胞与细胞外基质如纤维连接蛋白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.