| Innate immune cells recognize pathogen-associated molecule patterns (PAMPs) conserved in microbe by pattern recognition receptors (PRRs) to initiate host immune responses against infectious pathogens. CpG motif is abundant in bacteria DNA, and is methylated and suppressed in mammalian DNA. These differences between bacteria and mammalian host DNA make CpG motif function as PAMP to activate protective host immune responses. CpG oligodeoxynucleotides (CpG ODN) containing CpG motif can mimic the activity of bacterial DNA to activate immune cells, including B cells, NK cells, macrophages and dendritic cells, to produce nitric oxide (NO) and a variety of cytokines and chemokines. CpG ODN also induce maturation of dendritic cells and facilitate activation of Thl immune response. The adjuvant activity of CpG ODN has been proven useful in treatment of cancer, infectious and allergy diseases.CpG ODN recognition and signaling are mediated by a member of Toll-like receptor (TLR) family, namely TLR9. TLRs act as PRRs in detecting PAMPs and play important roles in triggering host defense immune responses. Upon recognizing respective ligand, TLR recruits adapter protein, myeloid differentiation protein 88 (MyD88), and activates mitogen-activated protein kinases (MAPKs) and nuclear factor-KB (NF-KB) through MyD88/TRAK/TRAF6/TAKl kinases cascade. Activation of both MAPK and NF-KB pathways is necessary for full activation of immune cells by TLR signaling.Small GTP-binding protein, Ras, is an important signal mediator in response to stimuli, such as growth factors, cytokines and hormones. In resting cells, Ras is maintained in inactivated Ras-GDP form. After activation, Ras is converted into active Ras-GTP. Ras exerts its function through activating downstream effectors, among which the best studied is Raf-MEK-ERK cascade. Although both CpG ODN and Ras can activate ERK, there is no report about the relevance of Ras in TLR9-mediated CpG ODN signal transduction. In this study, we investigated the role of Ras in CpG ODN signaling in murine macrophage cell line RAW264.7.Hongmei Xu, Dept. of Immunology, Second Military Medical University, Shanghai 200433 5To investigate the role of Ras protein in CpG ODN-induced macrophage activation, we observed the effects of FTI-277, a specific Ras inhibitor, on CpG ODN-induced NO and TNF-a production in murine macrophage cell line RAW264.7. Pretreatment with FTI-277 inhibited CpG ODN-induced NO and TNF-a production in a dose-dependent manner. Overexpression of a dominant-negative version of Ras, RasN17 (Serl7 is replaced with Asn), inhibited while wid-type Ras increased CpG ODN-induced NO and TNF-a production. Taken together, these results demonstrated that Ras activity is required for CpG ODN-induced NO and TNF-a production in macrophages.We then investigated the effects of wild-type Ras and RasN17 overexpression on CpG ODN-induced MAPK phosphorylation. Overexpression of wild-type Ras enhanced CpG ODN-induced ERK, JNK and NF-xJB reporter gene activation, On the contrary, RasN17 transfection inhibited CpG ODN-induced ERK1/2 and JNK1/2 phosphorylation and NF-KB reporter gene activation. These results demonstrated that Ras controls CpG ODN-induced ERK1/2 and JNK1/2 activation in macrophages. However, neither wild-type Ras nor dominant-negative Ras overexpression remarkably affects the activation of p38 kinase upon CpG ODN stimulation, suggesting p38 might be activated through a mechanism different from that of ERK and JNK. Overexpression of wild-type Ras and dominant-negative RasN17 also affect CpG ODN-induced IRAK1/TRAF6 complex formation, which suggest that Ras plays an important role in CpG ODN-induced IRAK1/TRAF6 complex formation in macrophages.Using Ras Activation Assay Kit, we found that CpG ODN activated Ras in a time-dependent manner, CpG ODN-induced Ras activation occurred within 2min, peaked at 5 min, and declined lOmin after CpG ODN treatment. Furthermore, CpG ODN-induced Ras activation was dose-dependent. The rapid activation of Ras follow...
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