ROLE OF PKD IN LEUKOCYTE ACTIVATION BY CPG DNA

描述(取自应用程序) 在某些类型的关节炎的滑液中可以发现细菌DNA 病人。细菌DNA(CpG DNA)中的CpG基序诱导白细胞产生 各种细胞因子，如肿瘤坏死因子-a，白介素6和白介素12，已被牵涉到 关节炎。通过细胞凋亡导致的细胞死亡被认为是维持 对自身抗原的免疫耐受，如果发生以下情况，可能会导致自身免疫性疾病 自身反应性免疫细胞不会发生凋亡。CpG DNA也保护B细胞 细胞不会发生凋亡。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诱导活性氧生成并激活 C-Jun氨基末端激酶(JNK)和p38。CpG DNA介导的ROS激活， JNK。P38可能参与了转录因子API和NFKB的激活 这导致随后的原癌基因表达和细胞因子的产生。 我们的初步数据表明，CpG DNA激活了蛋白激酶D 蛋白激酶C同工酶(PKD)，其活性被Go6976抑制 但不是Go6983。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。为了达到这一目标，我们将研究PKD在 CpG DNA介导的细胞因子产生与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 癌基因在WEHI-231和J774细胞中的表达及caspase的激活 WEHI-231细胞线粒体膜电位降低和细胞凋亡。 WEHI-231将引入显性活性PKD和显性负性PKD 利用可诱导逆转录病毒表达系统对J774细胞进行表达。逆转录病毒 转导细胞将通过流式细胞仪进行分析，电泳迁移率 移位试验、核糖核酸酶保护试验、体外激酶试验、蛋白质印迹、酶联免疫吸附试验 和共聚焦显微镜。PKD上游监管机构的候选人也将是 调查过了。这项拟议的研究将加深我们对CpG如何 细菌DNA中的基序破坏免疫耐受并与铬有关 炎症性自身免疫性疾病，如关节炎。

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.