RHO GTPASES IN ADHESION/CHEMOATTRACTANT RECEPTOR CROSST

在生理条件下，白细胞在细胞外基质中的氧化反应与分离的白细胞有很大的不同。在非贴壁细胞中，化学诱导剂和/或细胞因子能迅速刺激适度的氧化反应。相比之下，黏附的白细胞的反应表现出较长的延迟，随后氧代谢产物的生成显著增强和延长。黏附受体(整合素)和启动氧化反应的化学吸引剂受体之间的这种串扰的基础尚未确定。来自我们团队的大量数据表明，这种串扰发生在Rho GTP酶的水平上，Rho GTP酶控制着细胞骨架的整合素信号和化学诱导剂诱导的吞噬细胞NADPH氧化酶的激活。为了系统地研究黏附调节的氧化反应，我们将确定控制黏附的白细胞中Rho GTP酶功能和NADPH氧化酶激活的信号通路。我们将确定在贴壁细胞中NADPH氧化酶的组装和/或激活是否被修改。将使用完整的细胞系统和破碎的细胞系统来检测单个氧化酶调节蛋白的功能。在黏附调节的氧化反应过程中，Rho家族GTP酶的激活将使用新的生化分析方法进行测量。粘连引发的信号转导事件将被量化，并与GTPase活性和NADPH氧化酶活性的变化在药理学上相关联。将使用生化手段和遗传方法来研究特定信号通路的作用，在这些方法中，显性活性突变体或显性负性突变体将通过病毒表达系统引入细胞。最后，缺乏特定信号蛋白的转基因小鼠将被用来证实这些途径在调节黏附调节的氧化反应的组成部分中的作用。

The oxidative responses of leukocytes crawling through an extracellular matrix under physiological conditions differ dramatically from those of isolated leukocytes. In nonadherent cells, an oxidative response of modest extent is rapidly stimulated by chemoattractants and/or cytokines. In contrast, the response of adherent leukocytes exhibits a prolonged delay, followed by a dramatically enhanced and prolonged generation of oxygen metabolites. The basis for this crosstalk between adhesion receptors (integrins) and chemoattractant receptors that initiate the oxidative response has not been determined. Substantial data from our group suggests the hypothesis that this crosstalk occurs at the level of Rho GTPases which control both integrin signaling to the cytoskeleton and chemoattractant- induced activation of the phagocyte NADPH oxidase. In order to systematically investigate the adhesion-regulated oxidative response, we will determine the signaling pathways controlling Rho GTPase function and NADPH oxidase activation in adherent leukocytes. We will determine whether NADPH oxidase assembly and/or activation is modified in adherent cells. The function of individual oxidase regulatory proteins will be examined using intact cell and broken cell systems. The activation of Rho family GTPases will be measured during the adhesion-regulated oxidative response using novel biochemical assays. Signal transduction events initiated by adhesion will be quantified and correlated pharmacologically with changes in GTPase activity and NADPH oxidase activation. The role of specific signaling pathways will be examined using biochemical means and genetic approaches in which dominant active or dominant negative mutants will be introduced into cells using a viral expression system. Finally, transgenic mice lacking specific signaling proteins will be used to confirm the roles of these pathways in regulating components of the adhesion-regulated oxidative response.