The Rel/NF-κB family of transcription factors is induced in response to several signals. In unstimulated cells, NF-κB is associated in the cytoplasm with the inhibitory protein IκB. In response to an external signal, IκB is phosphorylated and degraded, releasing NF-κB to enter the nucleus and activate transcription [36
]. The wide variety of genes regulated by NF-κB includes cytokines, chemokines, adhesion molecules, acute-phase proteins, and inducible effector enzymes. The important role of NF-κB in the differentiation and activation of osteoclasts has been mentioned previously [38
]. Selective inhibition of NF-κB by several drugs blocks osteoclastogenesis [11
]. In the present study we have shown that selective inhibition of NF-κB with a ribbon-type NF-κB decoy could suppress the differentiation and activation of RANKL-induced osteoclastogenesis. Transfection of decoy ODN corresponding to the cis
sequences result in the attenuation of authentic cis-trans interaction, leading to the removal of trans
-factors from the endogenous cis
-element, with subsequent modulation of gene expression [39
]. The principle of the transcription factor decoy approach is based on the reduction of promoter activity as a result of the inhibition of binding of a transcription factor to a specific sequence in the promoter region. This approach is relatively simple and can be targeted to specific tissues; decoy ODN can be more effective than antisense ODN in blocking constitutively expressed factors as well as multiple transcription factors that bind to the same cis
]. However, one of the major limitations of the decoy ODN approach is the rapid degradation of phosphodiester ODN by intracellular nucleases [40
]. The lack of sequence specificity of phosphodiester ODN has been reported previously [29
]. To overcome these issues, the circular dumbbell double-stranded decoy ODN was developed [42
]. Circular dumbbell decoy ODN for AP-1 or E2F have been demonstrated to be more effective than conventional decoy ODN in previous studies [40
]. In this study, RNODN showed higher resistance to exonuclease and stronger binding activity on NF-κB than PNODN, and we examined the effect of RNODN for the inhibition of osteoclast differentiation and activation. A previous report [47
] showed the effect of decoy targeting NF-κB on apoptosis of human osteoclasts. In contrast to their results we were unable to show the specific effect of RNODN for apoptosis of rat osteoclasts. It is not yet clear whether NF-κB is responsible for the survival of osteoclasts [48
In this study, we were able to transfer decoy ODN to adherent macrophage/monocyte-like cells and osteoclast-like cells without reagent. The possibility and effectiveness of ODN transfer into these cells have been reported previously [49
]. The cellular uptake of ODN is reportedly achieved by a receptor-mediated endocytosis mechanism [50
]. However, the exact mechanism of cellular uptake of naked DNA or ODN is still poorly defined [52
]. The efficiency of internalizing naked DNA varies between cell types [52
]. In our study, the effectiveness of ODN transfer was promoted in serum-free conditions. The size of the ribbon-type decoy is about 20 base pairs, which is small compared with the plasmid, so it may be easier for ODN to be transferred into osteoclasts or their precursors.
In the pit formation assay of this study, we transferred the decoy on day 3. We were able to confirm TRAP-positive multinuclear cells on day 3 but the cells were not so large and it might be difficult to state that these cells were mature osteoclasts. It would have been better if we could have incubated mature osteoclasts on a hydroxyapatite-coated disc, but osteoclasts are easily damaged and it is technically difficult to subculture rat mature osteoclasts.
In the previous study, the gene encoding NFATc1, a member of the NFAT family of transcription factor genes, was found to be the most strongly induced transcription factor gene after stimulation by RANKL in osteoclast differentiation. NFATc1 autoamplifies its own gene, possibly by binding to its own promoter [35
]. The AP-1 and NF-κB binding sites are present with the promoter region of the NFATc1 gene [53
]. Recently, Takatsuna and colleagues showed that (-)-DHMEQ, a newly designed NF-κB inhibitor, inhibited RANKL-induced osteoclast differentiation in mouse bone marrow macrophages through the downregulation of NFATc1 [54
]. In the present study the expression of NFATc1 was inhibited by treatment with RNODN.
The skeletal complications of RA consist of focal bone erosions and periarticular osteoporosis at sites of active inflammation, and generalized bone loss with reduced bone mass. In rheumatoid synovium, activated T cells and fibroblasts express RANKL. TNF-α and IL-1β are also overproduced in synovium. TNF-α and IL-1 β, acting in concert with RANKL, can powerfully promote osteoclast recruitment, activation, and osteolysis in RA [55
]. In the synovium of patients with RA, NF-κB was present in most macrophages within the lining and sublining lesions throughout the synovium, including endothelial cells [56
]. CIA is an autoimmune model that in many ways resembles RA. Immunization of genetically susceptible rodents with type II collagen leads to the development of severe polyarticular arthritis mediated by an autoimmune response. Just as in RA, synovitis and erosions of cartilage and bone are hallmarks of CIA [58
]. In the present study, direct injection of RNODN in arthritic joints of rats with CIA led to an amelioration of arthritis and decreased the number of TRAP-positive cells in the synovium. The strategy of naked RNODN transfer into the joint implies a potential for future clinical treatment.