(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.
