ROLE OF PKD IN LEUKOCYTE ACTIVATION BY CPG DNA

PKD 在 CPG DNA 激活白细胞中的作用

基本信息

批准号：
6534528
负责人：
AE-KYUNG YI
金额：
$ 6.18万
依托单位：
UNIVERSITY OF TENNESSEE HEALTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-09-15 至 2003-08-31
项目状态：
已结题

项目摘要

DESCRIPTION (Taken from the application) Bacterial DNA can be found in the synovial fluid of some types of arthritis patients. CpG motifs in bacterial DNA (CpG DNA) induce leukocytes to produce various cytokines such as TNF-a, IL-6, and IL-12, which have been implicated in arthritis. Cell death by apoptosis is thought to be important in maintaining immune tolerance to self-antigens, and autoimmune disease may result if autoreactive immune cells fail to undergo apoptosis. CpG DNA also protects B cells from apoptosis. These data suggest that CpG DNA may be involved in the pathogenesis of arthritis and/or autoimmune diseases. Recently, we reported that CpG DNA induces reactive oxygen species (ROS) generation and activates c-Jun NH2 terminal kinase (JNK) and p38. CpG DNA-mediated activation of ROS, JNK. and p38 may contribute to activation of transcription factor API and NFKB which lead to the subsequent proto-oncogene expression and cytokine production. Our preliminary data demonstrated that CpG DNA activates protein kinase D (PKD), a protein kinase C (PKC) isoenzyme whose activity is inhibited by Go6976 but not by Go6983. The goal of this study is to understand CpG DNA-mediated signaling pathways which lead to cytokine production and B cell apoptosis protection. To approach this goal, we will investigate biologic role of PKD in the CpG DNA-mediated cytokine production and B cell apoptosis protection. We will evaluate the role of PKD on CpG DNA-mediated activation of signaling molecules and transcription factors (API and NFKB), cytokine production, and oncogene expression in WEHI-231 and J774 cells and caspase activation, mitochondrial membrane potential reduction, and apoptosis in WEHI-23 1 cells. Dominant active and dominant negative PKD will be introduced into WEHI-231 and J774 cells by using an inducible retroviral expression system. Retrovirally transduced cells will be analyzed by flow cytometry, electrophoretic mobility shift assay, RNase protection assay, in vitro kinase assay, western blot, ELISA and confocal microscopy. Candidate upstream regulators of PKD will also be investigated. This proposed study would enhance our understanding of how CpG motifs in bacterial DNA break immune tolerance and contribute to chrome inflammatory autoimmune diseases such as arthritis.

描述（取自应用程序）在某些类型的关节炎的滑液中可以找到细菌DNA 患者。细菌DNA（CpG DNA）中的CpG基序诱导白细胞产生各种细胞因子，例如TNF-A，IL-6和IL-12，与关节炎。凋亡对细胞死亡被认为对维持很重要如果如果自动反应性免疫细胞无法凋亡。 CpG DNA也保护B 细胞凋亡。这些数据表明CpG DNA可能参与关节炎和/或自身免疫性疾病的发病机理。最近，我们报道 CpG DNA诱导活性氧（ROS）生成并激活 C-Jun NH2末端激酶（JNK）和p38。 CpG DNA介导的ROS激活， jnk。 p38可能有助于转录因子API和NFKB的激活这导致了随后的原始癌基因表达和细胞因子的产生。我们的初步数据表明，CpG DNA激活蛋白激酶D （PKD），一种蛋白激酶C（PKC）同工酶，其活性被GO6976抑制但不是Go6983。这项研究的目的是了解CpG DNA介导的导致细胞因子产生和B细胞凋亡的信号通路保护。为了实现这一目标，我们将研究PKD的生物学作用 CPG DNA介导的细胞因子产生和B细胞凋亡保护。我们将评估PKD在CPG DNA介导的信号激活中的作用分子和转录因子（API和NFKB），细胞因子的产生以及 WEHI-231和J774细胞和caspase激活中的癌基因表达，线粒体膜的潜在减少和WEHI-23 1细胞的凋亡。主动和主导的负PKD将引入WEHI-231，并且 J774通过使用诱导病毒表达系统。逆转录病毒转导的细胞将通过流式细胞仪，电泳迁移率分析换档测定，RNase保护测定，体外激酶测定，蛋白质印迹，ELISA 和共聚焦显微镜。 PKD上游监管机构的候选人也将是调查。这项拟议的研究将增强我们对CPG的理解细菌DNA中断免疫耐受性的基序，并有助于铬炎症自身免疫性疾病，例如关节炎。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Role of vav1- and src-related tyrosine kinases in macrophage activation by CpG DNA.

vav1 和 src 相关酪氨酸激酶在 CpG DNA 激活巨噬细胞中的作用。

DOI：
10.1074/jbc.m311434200
发表时间：
2004
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Stovall,StephanieH;Yi,Ae-Kyung;Meals,ElizabethA;Talati,AjayJ;Godambe,SandipA;English,BKeith
通讯作者：
English,BKeith

Convergence of CpG DNA- and BCR-mediated signals at the c-Jun N-terminal kinase and NF-kappaB activation pathways: regulation by mitogen-activated protein kinases.