Bone Abstracts

CCN family member 2(CCN2)/Connective tissue growth factor (CTGF) is a multi-functional factor in proliferation, differentiation and migration of mesenchymal cells such as chondrocytes, osteoblasts and vascular endothelial cells. Recently, we also reported that CCN2/CTGF promotes osteoclastgenesis via induction of and interaction with DC-STAMP. We and other researchers reported that CCN2 binds to cytokines and receptors and modified their signaling. Therefore, we comprehensively sought additional factors binding to CCN2 and found receptor activator of NF-kappa B (RANK) as a potential CCN2-binding partner. Since RANK–RANK ligand (RANKL) signaling is indispensable for the osteoclastogenesis, we, in the present study, investigated the relation between CCN2 and RANK–RANKL system to clarify the role of CCN2 in the early stage of osteoclastogenesis.

We used a phage-display system to specify amino sequences binding to CCN2. Multiple molecular interactions were evaluated by solid-phase binding assays. The affinities of the bindings were calculated by using a surface plasmon resonance (SPR) analyzer. Nuclear translocation of NF-kappa B was monitored by fluorescence microscopy and was quantified using image analysis software. The activation of MAPKs was detected by western blotting. The differentiation of osteoclasts was monitored by TRAP staining.

CCN2 directly bound to RANK and enhanced the RANK-mediated signaling such as NF-kappa B, ERK and JNK pathways in pre-osteoclast cell RAW264.7. CCN2 did not affect on the binding of RANK–RANKL. CCN2 also bound to osteoprotegerin (OPG) via a common domain of CCN2. The affinity of CCN2 binding to OPG was comparable to that of RANKL to OPG. CCN2 suppressed the inhibitory effect of OPG on osteoclastogenesis. Of note, OPG inhibited the binding of CCN2 to RANK.

These findings indicate that CCN2 is a unique physiological factor, which can enhance osteoclastogenesis via interactions with both RANK and OPG in the early stage of osteoclastogenesis, in addition to stimulating the fusion of mononuclear osteoclast precursors.