The Vitamin D Receptor in Osteoblast-Lineage Cells Is Essential for the Proresorptive Activity of 1α,25(OH)2D3 In Vivo.

We previously reported that daily administration of a pharmacological dose of eldecalcitol, an analog of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], increased bone mass by suppressing bone resorption. These antiresorptive effects were found to be mediated by the vitamin D receptor (VDR) in osteoblast-lineage cells. Using osteoblast-lineage-specific VDR conditional knockout (Ob-VDR-cKO) mice, we examined whether proresorptive activity induced by the high-dose 1α,25(OH)2D3 was also mediated by VDR in osteoblast-lineage cells. Administration of 1α,25(OH)2D3 (5 µg/kg body weight/day) to wild-type mice for 4 days increased the number of osteoclasts in bone and serum concentrations of C-terminal crosslinked telopeptide of type I collagen (CTX-I, a bone resorption marker). The stimulation of bone resorption was concomitant with the increase in serum calcium (Ca) and fibroblast growth factor 23 (FGF23) levels, and decrease in body weight. This suggests that a toxic dose of 1α,25(OH)2D3 can induce bone resorption and hypercalcemia. In contrast, pretreatment of wild-type mice with neutralizing anti-receptor activator of NF-κB ligand (RANKL) antibody inhibited the 1α,25(OH)2D3-induced increase of osteoclast numbers in bone, and increase of CTX-I, Ca, and FGF23 levels in serum. The pretreatment with anti-RANKL antibody also inhibited the 1α,25(OH)2D3-induced decrease in body weight. Consistent with observations in mice conditioned with anti-RANKL antibody, the high-dose administration of 1α,25(OH)2D3 to Ob-VDR-cKO mice failed to significantly increase bone osteoclast numbers, serum CTX-I, Ca, or FGF23 levels, and failed to reduce the body weight. Taken together, this study demonstrated that the proresorptive, hypercalcemic, and toxic actions of high-dose 1α,25(OH)2D3 are mediated by VDR in osteoblast-lineage cells.

Effects of shokyo (Zingiberis Rhizoma) and kankyo (Zingiberis Processum Rhizoma) on prostaglandin E2 production in lipopolysaccharide-treated mouse macrophage RAW264.7 cells.

We previously reported that shokyo and kankyo, which are water-extracted fractions of ginger, reduced LPS-induced PGE2 production in human gingival fibroblasts. In this study, we examined the effects of these herbs on LPS-treated mouse macrophage RAW264.7 cells. Both shokyo and kankyo reduced LPS-induced PGE2 production in a concentration-dependent manner. Shokyo and kankyo did not inhibit cyclooxygenase (COX) activity, nor did they alter the expression of molecules in the arachidonic acid cascade. In addition, these herbs did not alter NF-κB p65 translocation into nucleus, or phosphorylation of p65 or ERK. These results suggest that shokyo and kankyo inhibit cPLA2 activity. Although 6-shogaol produced similar results to those of shokyo and kankyo, the concentration of 6-shogaol required for the reduction of PGE2 production were higher than those of 6-shogaol in shokyo and kankyo. Therefore, several gingerols and shogaols other than 6-shogaol may play a role in the reduction of LPS-induced PGE2 production. Thus, 6-shogaol, and other gingerols and shogaols inhibit cPLA2 activity and reduce LPS-induced PGE2 production via a different mechanism from traditional anti-inflammatory drugs. Moreover, kampo medicines that contain shokyo or kankyo are considered to be effective for inflammatory diseases.

Treatment of OPG-deficient mice with WP9QY, a RANKL-binding peptide, recovers alveolar bone loss by suppressing osteoclastogenesis and enhancing osteoblastogenesis.

Osteoblasts express two key molecules for osteoclast differentiation, receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG), a soluble decoy receptor for RANKL. RANKL induces osteoclastogenesis, while OPG inhibits it by blocking the binding of RANKL to RANK, a cellular receptor of RANKL. OPG-deficient (OPG-/-) mice exhibit severe alveolar bone loss with enhanced bone resorption. WP9QY (W9) peptide binds to RANKL and blocks RANKL-induced osteoclastogenesis. W9 is also reported to stimulate bone formation in vivo. Here, we show that treatment with W9 restores alveolar bone loss in OPG-/-mice by suppressing osteoclastogenesis and enhancing osteoblastogenesis. Administration of W9 or risedronate, a bisphosphonate, to OPG-/-mice significantly decreased the osteoclast number in the alveolar bone. Interestingly, treatment with W9, but not risedronate, enhanced Wnt/ß-catenin signaling and induced alveolar bone formation in OPG-/-mice. Expression of sclerostin, an inhibitor of Wnt/ß-catenin signaling, was significantly lower in tibiae of OPG-/-mice than in wild-type mice. Treatment with risedronate recovered sclerostin expression in OPG-/-mice, while W9 treatment further suppressed sclerostin expression. Histomorphometric analysis confirmed that bone formation-related parameters in OPG-/-mice, such as osteoblast number, osteoblast surface and osteoid surface, were increased by W9 administration but not by risedronate administration. These results suggest that treatment of OPG-/-mice with W9 suppressed osteoclastogenesis by inhibiting RANKL signaling and enhanced osteoblastogenesis by attenuating sclerostin expression in the alveolar bone. Taken together, W9 may be a useful drug to prevent alveolar bone loss in periodontitis.

Evaluation of pharmaceuticals with a novel 50-hour animal model of bone loss.

Osteoporosis remains a major public health problem through its associated fragility fractures. Several animal models for the study of osteoporotic bone loss, such as ovariectomy (OVX) and denervation, require surgical skills and several weeks to establish. Osteoclast differentiation and activation is mediated by RANKL. Here we report the establishment of a novel and rapid bone loss model by the administration of soluble RANKL (sRANKL) to mice. Mice were injected intraperitoneally with sRANKL and used to evaluate existing anti-osteoporosis drugs. sRANKL decreased BMD within 50 h in a dose-dependent manner. The marked decrease in femoral trabecular BMD shown by pQCT and the 3D images obtained by microCT were indistinguishable from those observed in the OVX model. Histomorphometry showed that osteoclastic activity was significantly increased in the sRANKL-injected mice. In addition, serum biochemical markers of bone turnover such as Ca, C-telopeptide of type 1 collagen (CTX), and TRACP5b were also significantly increased in the sRANKL-injected mice in a dose-dependent manner. Bisphosphonates (BPs), selective estrogen receptor modulators (SERMs), and PTH are commonly used for the treatment of osteoporosis. We successfully evaluated the effects of anti-bone-resorbing agents such as BPs, a SERM, and anti-RANKL-neutralizing antibody on bone resorption in a couple of weeks. We also evaluated the effects of PTH on bone formation in 2 wk. A combination of sRANKL injections and OVX made it possible to evaluate a SERM. The sRANKL model is the simplest, fastest, and easiest of all osteoporosis models and could be useful in the evaluation of drug candidates for osteoporosis.

Identification of the functional domain of osteoclast inhibitory peptide-1/hSca.

Osteoclast (OCL) activity is controlled by local factors produced in the bone microenvironment. We previously identified a novel inhibitor of OCL formation that is produced by OCLs (osteoclast inhibitory peptide-1/human Sca [OIP-1/hSca]). OIP-1/hSca is a glycosylphosphatidylinositol (GPI)-linked membrane protein (16 kDa) that is cleaved from the OCL surface. Immunocytochemical staining further confirmed the expression of OIP-1/hSca in OCL formed in mouse bone marrow cultures. However, the structure/function mechanisms responsible for the inhibitory effects of OIP-1/hSca on OCL formation are unknown. Therefore, we expressed deletion mutants of OIP-1 in 293 cells and tested their effects on OCL formation. These studies indicated that the carboxy-terminal peptide (c-peptide) region is critical for OIP-1/hSca activity. A 33 amino acid OIP-1 c-peptide (10-100 ng/ml) significantly inhibited 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced OCL formation and pit formation capacity of OCL on dentine slices in human bone marrow cultures. Furthermore, the c-peptide (10-100 ng/ml) significantly inhibited early human OCL precursor (granulocyte-macrophage colony-forming unit [GM-CFU]) colony formation in methylcellulose cultures. The polyclonal antibody against the OIP-1 c-peptide neutralized the inhibitory effect of OIP-1 c-peptide on OCL formation in mouse bone marrow cultures in vitro. These results show that the OIP-1 c-peptide is the functional domain of OIP-1 and that availability of neutralizing antibody specific to the OIP-1 c-peptide should provide important mechanistic insights into OIP-1/hSca inhibition of osteoclastogenesis in the bone microenvironment.