Osteoclasts (OCs) are multinucleated cells derived from circulating osteoclast precursor cells (OCPs) of the monocyte/macrophage lineage. They represent the only cell type capable of bone resorption [5
]. Osteoclasts promote bone resorption in metabolic, degenerative and neoplastic bone disorders. Excess osteoclast activity in osteolysis may involve not only generation and activation of OCs, but also increased recruitment of OCPs. The increased bone resorption in osteolysis is thought to be mediated via numerous pro inflammatory cytokines. These cytokines are considered to act primarily via a common final pathway involving members of the TNF receptor-ligand family: The RANKL and its corresponding RANK receptor that play a crucial role in osteoclast differentiation and activation, and OPG, the physiological inhibitor of RANKL [15
]. In turn, excess osteoclast activity is associated with the disorders of the TNF receptor-ligand family that ultimately lead to metabolic, degenerative and neoplastic bone disorders. Clinical treatments are needed that can inhibit excess osteolysis in an inflammatory microenvironment. Given that RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis, we tested the hypotheses that inhibition of RANK expression by RNA silencing would reduce the number of osteoclasts and the activity of bone erosion.
The limitation of our study is that BMMs culture experiments have a limited perspective of time as we analyzed the BMM cells for only nine days. As metabolic, degenerative and neoplastic bone disorders are processes which can take years, the reactions by osteoclasts might change in the course of time. The correlational research in vivo should be carried on in future studies. Nevertheless, we demonstrated that the difference in osteoclast number and bone erosion between the study groups was significant.
Our data showing inhibition of RANK expression by successful silencing of RNA, which can inhibit specific gene expression, suggests that we can obtain the optimal shRNA-silencing RANK. The inhibition rates of pshRANK-1, pshRANK-2 and pshRANK-3 by Western Blot were 59.4%, 63.3% and 88.3% (p < 0.01) respectively compared with the control plasmid pSUPER-retro-puro, which is consistent with the result of Real-time PCR. Thus, shRANK-3 was identified as the optimal sequence silencing RANK.
Our study shows that inhibition of RANK expression suppresses osteoclast differentiation, thus leading to reduction of bone resorption. We found that RANK gene-silencing using retrovirus-mediated shRNA can significantly suppress RANK expression of BMMs. The inhibition rates of RANK protein by Western Blot was about 80.7% (p < 0.01). TRAP staining and SEM reveal that the osteoclastogenesis of infected BMMs is significantly reduced, compared with uninfected BMMs.
We are not aware of any study showing a similar effect of retrovirus-mediated gene therapy with pshRANK on osteoclastogenesis in BMMs. Although OPG fusion protein and OPG have been used successfully to prevent osteolysis, OPG may bind TNF related apoptosis-inducing ligand in addition to RANKL and thus act as a cancer survival factor [16
]. Clinical trials with a monoclonal antibody against RANKL showed efficacy and anti-resorptive activity [19
], but sensitization to monoclonal antibodies` therapeutics poses significant risk to the patient and may blunt the efficacy of these therapies [20
]. In addition, antibodies against OPG fusion protein harbor the potential risk of cross-reacting with and neutralizing endogenous OPG.
Numerous studies have shown that the RANK/RANKL pathway is central to cellular and molecular mechanisms associated with the process of osteolysis [4
Subsequently, a gene-silenced RANK gene leads to an imbalance of a variety of downstream signaling pathways which are required for osteoclast development [21
]. RANKL binding to RANK induces the trimerization of RANK and TNF receptor-associated factors 6 (TRAF6), which leads to the activation of mitogen-activated kinases (MAPK). TRAF6 adaptor protein binds to the intracellular part of the activated RANK receptor and starts the NF-κB pathway, which finally leads to activation of osteoclastogenesis [22
]. Different members of the MAPK family, such as p38-MAPKα and p38-MAPKβ, are known to be involved in osteoclastogenesis [23
]. Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis is mediated by receptor activator of NF-κB ligand (RANKL) [23
]. Furthermore, an influence on bone metabolism is caused by the induction of the phoshatidylinositol (PI) metabolism, which is mediated by the c-Src kinase. The targeted disruption of the c-Src proto-oncogene leads to osteopetrosis in mice [24
]. Following notable advances in the understanding of intracellular signaling pathways of RANK, therapeutic drugs specifically targeting downstream signaling molecules have been developed including Interferon-β, γ, p38 inhibitor (SB203580, FR167653), JNK inhibitor (SB600125), IKappaB kinase (IKK) and NEMO binding domain (NBD peptide) inhibitor, NF-κB inhibitor (NF-κB decoy), Calcineurin inhibitor (Cyclosporin A, FK 506), NFAT inhibitor (VIVIT peptide), PI3K inhibitor (Wortmannin, LY290442) [25
]. The limitation of these approaches lies in the lack of specificity of these compounds, since these intracellular signaling pathways are also important for the function and homeostasis of other cells and tissues. Furthermore, RANK is expressed by other types of cells (inflammatory cells and endothelial cells) and plays a role in other physiological processes such as inflammation and angiogenesis. This requires the development of a sophisticated cell-specific or tissue-specific drug delivery system to prevent or mitigate their adverse effects [31
RANK suppression in the RANKL/RANK system appears to be a promising target for potential therapies in the treatment of osteolysis. RNA interference to inhibit specific gene expression has been shown to be an effective and promising technology for both basic science research and therapeutic intervention [32