Tetracycline, an Appropriate Reagent for Measuring Bone-Formation Activity in the Murine Model of the Streptococcus mutans-Induced Bone Loss.

Tetracycline is used as a fluorescent reagent to measure bone formation activity in bone histomorphometric analyses. However, there is a possibility to lead a different conclusion when it is used in a bacteria-infected murine model since the tetracycline is considered to work as an antibiotic reagent. There are non-antibiotic fluorescent reagents such as alizarin and calcein for measuring bone formation activity. The purpose of this study was to clarify whether tetracycline could be an appropriate reagent to measure bone formation activity in a murine bacterial model in the same way as a non-antibiotic fluorescent reagent. We used Streptococcus mutans (S. mutans), a normal inhabitant in the oral cavity and tetracycline-sensitive bacteria, for inducing the bacterial model. The murine bacterial model was generated by intravenously inoculating S. mutans to the tail vein, followed immediately by the injection of the first fluorescent reagent, and the second one was injected 2 days prior to euthanization. After one day of inoculation with S. mutans, the subcutaneously injected alizarin had a similar colony count derived from the liver and the bone marrow tissue compared to the phosphate buffered saline (PBS)-injected control group. On the other hand, subcutaneous injection of tetracycline led to a significantly lower colony count from the liver compared to alizarin- or calcein-injected group. However, on day seven, after S. mutans intravenous injections, bone mineral density of distal femurs was significantly reduced by the bacteria inoculation regardless of which fluorescent reagents were injected subcutaneously. Finally, S. mutans inoculation reduced bone-formation-activity indices in both the tetracycline-alizarin double-injected mice and the calcein-alizarin double-injected mice. These results suggested that a one-time injection of tetracycline did not affect bone formation indices in the S. mutans-induced bone loss model. Tetracycline could be used for measuring bone formation activity in the same way as non-antibiotic fluorescent reagent such as calcein and alizarin, even in a tetracycline-sensitive bacterium-infected model.

The Effects of Receptor Activator of NF-κB Ligand-Binding Peptides on Bone Resorption and Bone Formation.

Receptor activator of NF-κB ligand (RANKL)-binding peptides inhibit bone resorption and were recently shown to activate bone formation. The stimulatory mechanism underlying bone formation associated with these peptides was explained as RANKL-reverse signaling, wherein RANKL molecules on osteoblasts work as receptors to stimulate osteoblast differentiation. However, why RANKL-binding peptides stimulate osteoblast differentiation while osteoprotegerin (OPG), which is well known to bind to RANKL, cannot activate osteoblast differentiation has remained unclear. In this mini-review, we introduce three main issues: (1) The inhibitory effects of two RANKL-binding peptides (W9 and OP3-4) on bone resorption; (2) The stimulatory effects of the RANKL-binding peptides on osteoblast differentiation; and (3) The accumulation and membrane clustering of RANKL molecules at the cell surface of osteoblasts as a potential molecular switch stimulating osteoblast differentiation by RANKL-binding peptides.

A novel inhibitor of NF-κB-inducing kinase prevents bone loss by inhibiting osteoclastic bone resorption in ovariectomized mice.

Musculoskeletal diseases and disorders, including osteoporosis and rheumatoid arthritis are diseases that threaten a healthy life expectancy, and in order to extend the healthy life expectancy of elderly people, it is important to prevent bone and joint diseases and disorders. We previously reported that alymphoplasia (aly/aly) mice, which have a loss-of-function mutation in the Nik gene involved in the processing of p100 to p52 in the alternative NF-κB pathway, show mild osteopetrosis with a decrease in the osteoclast number, suggesting that the alternative NF-κB pathway is a potential drug target for ameliorating bone diseases. Recently, the novel NF-κB-inducing kinase (NIK)-specific inhibitor compound 33 (Cpd33) was developed, and we examined its effect on osteoclastic bone resorption in vitro and in vivo. Cpd33 inhibited the receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis accompanied by a decrease in the expression of nfatc1, dc-stamp, and cathepsin K, markers of osteoclast differentiation, without affecting the cell viability, in a dose-dependent manner. Cdp33 specifically suppressed the RANKL-induced processing of p100 to p52 but not the phosphorylation of p65 or the degradation or resynthesis of IκBα in osteoclast precursors. Cpd33 also suppressed the bone-resorbing activity in mature osteoclasts. Furthermore, Cdp33 treatment prevented bone loss by suppressing the osteoclast formation without affecting the osteoblastic bone formation in ovariectomized mice. Taken together, NIK inhibitors may be a new option for patients with a reduced response to conventional pharmacotherapy or who have serious side effects.

Bif-1/Endophilin B1/SH3GLB1 regulates bone homeostasis.

Skeletal tissue homeostasis is maintained via the balance of osteoclastic bone resorption and osteoblastic bone formation. Autophagy and apoptosis are essential for the maintenance of homeostasis and normal development in cells and tissues. We found that Bax-interacting factor 1 (Bif-1/Endophillin B1/SH3GLB1), involving in autophagy and apoptosis, was upregulated during osteoclastogenesis. Furthermore, mature osteoclasts expressed Bif-1 in the cytosol, particularly the perinuclear regions and podosome, suggesting that Bif-1 regulates osteoclastic bone resorption. Bif-1-deficient (Bif-1 -/- ) mice showed increased trabecular bone volume and trabecular number. Histological analyses indicated that the osteoclast numbers increased in Bif-1 -/- mice. Consistent with the in vivo results, osteoclastogenesis induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) was accelerated in Bif-1 -/- mice without affecting RANKL-induced activation of RANK downstream signals, such as NF-κB and mitogen-activated protein kinases (MAPKs), CD115/RANK expression in osteoclast precursors, osteoclastic bone-resorbing activity and the survival rate. Unexpectedly, both the bone formation rate and osteoblast surface substantially increased in Bif-1 -/- mice. Treatment with ß-glycerophosphate (ß-GP) and ascorbic acid (A.A) enhanced osteoblastic differentiation and mineralization in Bif-1 -/- mice. Finally, bone marrow cells from Bif-1 -/- mice showed a significantly higher colony-forming efficacy by the treatment with or without ß-GP and A.A than cells from wild-type (WT) mice, suggesting that cells from Bif-1 -/- mice had higher clonogenicity and self-renewal activity than those from WT mice. In summary, Bif-1 might regulate bone homeostasis by controlling the differentiation and function of both osteoclasts and osteoblasts (235 words).