Exogenous regucalcin stimulates osteoclastogenesis and suppresses osteoblastogenesis through NF-κB activation

Regucalcin plays a pivotal role in regulating intracellular calcium homeostasis and consequently has a profound effect on multiple intracellular signal transduction pathways. The regucalcin transgenic rat displays pronounced bone loss, and bone marrow from these animals exhibits significantly elevated osteoclast formation. Consistent with these effects exogenous regucalcin promotes osteoclastogenesis in mouse bone marrow cultures, but interestingly regucalcin suppresses the differentiation and mineralization of MC3T3 osteoblast precursors. However, the molecular mechanisms involved are presently unclear. As the nuclear factor-kappa B (NF-κB) signal transduction pathway is critical to osteoclastogenesis but inhibitory of osteoblastogenesis, we hypothesized that regucalcin may promote osteoclastogenesis and suppress osteoblastogenesis upregulating NF-κB signal transduction. In this study, we examined the effect of regucalcin on receptor activator of NF-κB (RANK) ligand (RANKL) -induced osteoclast formation using the RAW264.7 monocytic cell line and osteoblast formation using the pre-osteoblastic cell line MC3T3. As expected, culture with exogenous regucalcin was found to enhance RANKL-induced osteoclastogenesis. Consistent with this effect regucalcin increased basal and RANKL-induced NF-κB activation as assessed by NF-κB luciferase assay. The capacity of regucalcin to augment RANKL-induced NF-κB activity was inhibited by menaquinone-7, a potent NF-κB antagonist, while the Erk inhibitor PD98059 and staurosporine had no effect, demonstrating a specific effect on NF-κB signaling. By contrast, regucalcin inhibited mineralization of MC3T3 cells and enhanced tumor necrosis factor-α (TNFα)-induced NF-κB activation. As with NF-κB induction in osteoclasts, NF-κB activation was abolished by addition of the NF-κB antagonist menaquinone-7, but not by PD98059 and staurosporine. Transforming growth factor-β (TGFβ) and bone morphogenic protein-2 (BMP2) are potent early commitment and late osteoblast differentiation factors, respectively, and both mediate their actions through the Smad-signal transduction pathway, a system that is extremely sensitive to and inhibited by TNFα-induced NF-κB. We consequently examined the effect of regucalcin on TGFβ and BMP2-induced Smad activation in the presence and absence of TNFα. While regucalcin had no effect on basal Smad activation by TGFβ and BMP2, it enhanced the suppressive effect of TNFα on both TGFβ- and BMP2-induced Smad activations. Taken together, present data suggest that regucalcin may induce bone loss in vivo by promoting osteoclasts and simultaneously suppressing osteoblasts through amplification of basal and/or cytokine-induced NF-κB activation. Regucalcin may have a role as a modulator in NF-κB activation.