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Schlafen-2 (slfn-2) is a member of slfn family, regulators of T cell development and its expression is altered during infection by microbial pathogens. However, the molecular mechanism involved in slfn expression is still to be determined. In this study, we isolated slfn-2 as a LPS-induced differentially expressed genes (DEGs) in RAW 264.7 cells and examined expression and regulation of slfn-2 in CpG-DNA-treated and LPS-treated macrophages. We defined a transcriptional start site in the slfn-2 gene. To examine the promoter organization of the slfn-2 gene, we cloned a approximately 1.8 kb region upstream of the transcription start site. Sequence analysis indicates consensus sites for AP-1 and NF-kappaB. Comprehensive mutant analyses, ELISA-based transcription factor activation assay, and ChIP assays reveal that functional interaction of AP-1 and NF-kappaB with the promoter element is necessary for the Toll-like receptor (TLR)-mediated slfn-2 gene expression by CpG-DNA and LPS treatment in macrophages. In summary, we identified a slfn-2 promoter for the first time and demonstrated that CpG-DNA and LPS triggers slfn-2 gene expression by activating NF-kappaB and AP-1 pathways in macrophages.

Synthetic oligodeoxynucleotides (CpG-ODNs) and bacterial DNA containing unmethylated CpG dinucleotides in the context of particular base sequences (CpG motifs) are known to inhibit anti-IgM-induced growth arrest and apoptosis of WHEI 231 B lymphocytes, and spontaneous apoptosis of mature spleen B cells in a sequence-specific fashion of the CpG-ODN. Here we report that CpG-ODN protects from the cell death induced by γ-irradiation of primary mouse spleen cells as well as mouse RAW 264.7 macrophage cells and human RPMI 8226 B cells. Experimental results showed that CpG-ODN promotes growth of the cells, and protects the cells from γ-irradiation-induced cell death accompanying Bcl-xS/L and Bcl-2 upregulation. Furthermore, survival of macrophages was enhanced when splenocytes were pretreated with CpG-ODN. Our results suggest the potential application of CpG-ODNs for more efficient cancer radiotherapy by enhancing survival of normal immune cells after radiation damage.

Pyrrolidine dithiocarbamate (PDTC) is a stable compound that acts as antioxidant or prooxidant, and is widely used to inhibit the activation of NF-kappaB. PDTC was also reported to activate NF-kappaB depending on its dose and metal ions in PC12 cells. In this work, we demonstrated a working mechanism of PDTC and its effects on the proinflammatory cytokine gene expression in a mouse macrophage cell line, RAW 264.7. PDTC alone induced NF-kappaB-independent MIP-2 promoter activation that can be assessed by transient transfection and confocal image analysis. The involvement of AP-1 transcription factor was noticed by promoter deletion/site-specific mutation analysis and electrophoretic mobility shift assay (EMSA). Among three different mitogen-activated protein kinase (MAPK) pathways tested, only the stress-activated protein kinase (SAPK)/Jun N-terminal kinase (JNK) pathway was significantly activated in RAW 264.7 cells after the stimulation with PDTC. Using pathway-specific inhibitors, we found that the SAPK/JNK pathway is clearly associated with PDTC-induced MIP-2 gene expression. Our experimental results indicate that PDTC-induced proinflammatory cytokine expressions are mediated by SAPK/JNK pathway, which activates AP-1.

Matrix metalloproteinase-9 (MMP-9) is a secreted type IV collagenase that plays an important role in the remodeling of the extracellular matrix (ECM) and the migration of normal and tumor cells. We have shown that CpG-ODN-induced migration of RAW 264.7 cell is regulated by MMP-9 activity by using tissue inhibitors of MMP-1 (TIMP-1). The MMP-9 gene expression was transcriptionally induced by CpG-ODN in a time-dependent manner. An MMP-9 promoter-reporter was activated by the stimulation of CpG-ODN and ectopical expression of NF-kappaB transcription factor. Inhibition of NF-kappaB nuclear localization by co-expression of a mutant IkappaBalpha protein blocked the CpG-ODN-induced MMP-9 promoter activation. BMS-345541, an IKK-2 inhibitor also inhibited the expression of MMP-9 gene induced by CpG-ODN. Direct binding of NF-kappaB protein to the promoter region of the MMP-9 was confirmed by chromatin immunoprecipitation using NF-kappaB antibody. These results lead us to a conclusion that NF-kappaB activation is required for MMP-9 gene expression. In summary, our data suggest that NF-kappaB-dependent expression of MMP-9 in response to CpG-ODN plays an important role in the recruitment of immune cells.