Bone is constantly remodeled by the activities of osteoclasts and osteoblasts. Osteoclasts break down the extracellular matrix of bone, while osteoblasts create it. These activities are balanced in youth, but with advancing age a range of mechanisms operate to create a growing imbalance favoring osteoclasts. This steadily reduces bone density leading to osteoporosis and eventually life-threatening fracture risk. In principle any compensatory therapy should be beneficial, any way to suppress osteoclast or enhance osteoblast populations and activity regardless of whether or not underlying causes are targeted. In practice, finding good paths forward has been challenging, but researchers here report on their investigation of one potential new approach.
A CpG oligodeoxynucleotide (CpG-ODN), iSN40, was originally identified as promoting the mineralization and differentiation of osteoblasts, independent of Toll-like receptor 9 (TLR9). Since CpG ODNs are often recognized by TLR9 and inhibit osteoclastogenesis, this study investigated the TLR9 dependence and anti-osteoclastogenic effect of iSN40 to validate its potential as an osteoporosis drug.
The murine monocyte/macrophage cell line RAW264.7 was treated with the receptor activator of nuclear factor-κB ligand (RANKL) to induce osteoclast differentiation, then the effect of iSN40 on was 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 and inducing anti-/non-osteoclastogenic genes. Treatment with a TLR9 inhibitor or a mutation in the CpG motif of iSN40 abolished the intracellular uptake and anti-osteoclastogenic effect of iSN40.
These results demonstrate that iSN40 is subcellularly internalized and is recognized by TLR9 via its CpG motif, modulates RANKL-dependent osteoclastogenic gene expression, and ultimately inhibits osteoclastogenesis. Finally, iSN40 was confirmed to inhibit the osteoclastogenesis of RAW264.7 cells cocultured with the murine osteoblast cell line MC3T3-E1, presenting 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.
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