Osteogenic CpG oligodeoxynucleotide, iSN40, inhibits osteoclastogenesis in a TLR9-dependent manner
A CpG oligodeoxynucleotide (CpG-ODN), iSN40 (5-GGA ACG ATC CTC AAG CTT-3), was originally identified to promote osteoblast differentiation independent of Toll-like receptor 9 (TLR9). While CpG-ODNs are generally known to be recognized by TLR9 and inhibit osteoclasto-genesis. This study investigated the anti-osteoclastogenic effect of iSN40. The murine mono-cyte/macrophage cell line RAW264.7 was treated with receptor activator of nuclear factor-{kappa}B ligand (RANKL) to induce osteoclast differentiation, and the effects of iSN40 on osteoclast formation were quantified by tartrate-resistant acid phosphatase (TRAP) staining and real-time RT-PCR. iSN40 completely inhibited RANKL-induced differentiation into TRAP+ multinucleated osteoclasts by suppressing osteoclastogenic genes (Nfatc1, Ctsk, and Dcstamp) and inducing anti-/non-osteoclasto-genic genes (Irf8, Adgre1, and Il1b). Treatment with a TLR9 inhibitor, E6446, or mutation in the CpG motif of iSN40 abolished intracellular uptake and the anti-osteoclastogenic effect of iSN40. These results demonstrate that iSN40 is internalized subcellularly, recognized by TLR9 via its CpG motif, modulates RANKL-dependent osteoclastogenic gene expression, and ultimately inhibits osteoclast formation. Computational simulation of the iSN40 structure also suggested the importance of the superficial CpG motif for iSN40 function. Finally, iSN40 was confirmed to inhibit osteoclastogenesis of RAW264.7 cells cocultured with the murine osteoblast cell line MC3T3-E1, which is a model of bone remodeling. This study demonstrates that iSN40, which exerts both pro-osteogenic and anti-osteoclastogenic effects, may be a promising nucleic acid drug for osteoporosis.