ROLE OF PKD IN LEUKOCYTE ACTIVATION BY CPG DNA

PKD 在 CPG DNA 激活白细胞中的作用

• 批准号: 6344447
• 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
• 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
• 来源: https://reporter.nih.gov/project-details/6344447
• 关键词: B lymphocyte; CpG islands; JAK kinase; RNase protection assay; apoptosis; autoimmune disorder; bacterial DNA; biological signal transduction; confocal scanning microscopy; enzyme activity; enzyme linked immunosorbent assay; flow cytometry; gel mobility shift assay; gene expression; immune tolerance /unresponsiveness; leukocyte activation /transformation; phosphorylation; protein kinase; protein kinase C; protein structure function; transcription factor; western blottings

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） 在某些类型关节炎的滑液中可以发现细菌DNA 患者细菌DNA中的CpG基序（CpG DNA）诱导白细胞产生 各种细胞因子，如TNF-α、IL-6和IL-12，它们已经涉及 关节炎细胞凋亡导致的细胞死亡被认为是维持细胞凋亡的重要因素。 对自身抗原的免疫耐受，如果 自身反应性免疫细胞不能经历凋亡。CpG DNA也保护B 细胞凋亡。这些数据表明，CpG DNA可能参与了 关节炎和/或自身免疫性疾病的发病机制。最近，我们报道了 CpG DNA诱导活性氧（ROS）的产生， c-Jun NH 2末端激酶（JNK）和p38。CpG DNA介导的ROS活化， JNK。p38可能参与转录因子API和NF κ B的激活 其导致随后的原癌基因表达和细胞因子产生。 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Go 6976抑制 而不是Go 6983。本研究的目的是了解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在以下方面的生物学作用： 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对CpG DNA介导的信号转导激活的作用 分子和转录因子（API和NF κ B），细胞因子产生，和 WEHI-231和J774细胞中的癌基因表达和半胱天冬酶激活， WEHI-23 - 1细胞线粒体膜电位降低和凋亡。 将显性活性和显性阴性PKD导入WEHI-231， J774细胞中表达。逆转录病毒地 转导的细胞将通过流式细胞术、电泳迁移率 转移试验，RNA酶保护试验，体外激酶试验，蛋白质印迹，ELISA 和共聚焦显微镜。PKD的候选上游调节剂也将是 研究了这项拟议的研究将加强我们对CpG如何 细菌DNA中的基序破坏免疫耐受并促进Chrome 炎症性自身免疫性疾病，如关节炎。