Sclerostin deficiency effectively promotes bone morphogenetic protein-2-induced ectopic bone formation.

BACKGROUND AND OBJECTIVE: Severe periodontitis causes alveolar bone resorption, resulting in tooth loss. Developments of tissue regeneration therapy that can restore alveolar bone mass are desired for periodontal disease. The application of bone morphogenetic protein-2 (BMP-2) has been attempted for bone fractures and severe alveolar bone loss. BMP-2 reportedly induces sclerostin expression, an inhibitor of Wnt signals, that attenuates bone acquisition. However, the effect of sclerostin-deficiency on BMP-2-induced bone regeneration has not been fully elucidated. We investigated BMP-2-induced ectopic bones in Sost-knockout (KO) mice. METHODS: rhBMP-2 were implanted into the thighs of C57BL/6 (WT) and Sost-KO male mice at 8 weeks of age. The BMP-2-induced ectopic bones in these mice were examined on days 14 and 28 after implantation. RESULTS: Immunohistochemical and quantitative RT-PCR analyses showed that BMP-2-induced ectopic bones expressed sclerostin in osteocytes on days 14 and 28 after implantation in Sost-Green reporter mice. Micro-computed tomography analysis revealed that BMP-2-induced ectopic bones in Sost-KO mice showed a significant increased relative bone volume and bone mineral density (WT = 468 mg/cm3 , Sost-KO = 602 mg/cm3 ) compared with those in WT mice on day 14 after implantation. BMP-2-induced ectopic bones in Sost-KO mice showed an increased horizontal cross-sectional bone area on day 28 after implantation. Immunohistochemical staining showed that BMP-2-induced ectopic bones in Sost-KO mice had an increased number of osteoblasts with osterix-positive nuclei compared with those in WT mice on days 14 and 28 after implantation. CONCLUSION: Sclerostin deficiency increased bone mineral density in BMP-2-induced ectopic bones.

Sclerostin blockade promotes bone metastases of Wnt-responsive breast cancer cells.

The secreted protein sclerostin is primarily produced by osteocytes and suppresses osteoblast differentiation and function by inhibiting the canonical Wnt signaling pathway. Genetic and pharmacological inhibition of sclerostin has been shown to increase bone formation and an anti-sclerostin antibody has been clinically approved for the treatment of osteoporosis. Canonical Wnt signaling is also involved in the progression of several types of cancers including breast cancer. Here, we studied the effects of sclerostin inhibition on the development of bone metastases of breast cancer using mouse models. TOPFLASH assay and real-time PCR analysis of AXIN2, a target of canonical Wnt signaling, revealed that, among four cell lines tested, MDA-MB-231 human breast cancer cells responded highly to the canonical Wnt ligand Wnt3a, whereas other cell lines exhibited marginal responses. Consistent with these results, treatment with an anti-sclerostin antibody significantly increased the bone metastases of MDA-MB-231 but not those of other breast cancer cells. Immunohistochemical studies demonstrated that an anti-sclerostin antibody induced intracellular accumulation of ß-catenin in bone-colonized MDA-MB-231 cells. Suspension culture assays showed that Wnt3a accelerated the tumorsphere formation of MDA-MB-231 cells, whereas monolayer cell proliferation and migration were not affected. Furthermore, the numbers of osteoclasts and their precursor cells in bone metastases of MDA-MB-231 were significantly increased in mice treated with an anti-sclerostin antibody. These results collectively suggest that sclerostin blockade activates canonical Wnt signaling in ligand-responsive breast cancer cells metastasized to bone, thereby increasing bone metastases, likely to have been mediated at least in part by enhancing stem cell-like properties of cancer cells and osteoclastogenesis.

Positive and Negative Regulators of Sclerostin Expression.

Sclerostin is secreted from osteocytes, binds to the Wnt co-receptor Lrp5/6, and affects the interaction between Wnt ligands and Lrp5/6, which inhibits Wnt/ß-catenin signals and suppresses bone formation. Sclerostin plays an important role in the preservation of bone mass by functioning as a negative regulator of bone formation. A sclerostin deficiency causes sclerosteosis, which is characterized by an excess bone mass with enhanced bone formation in humans and mice. The expression of sclerostin is positively and negatively regulated by many factors, which also govern bone metabolism. Positive and negative regulators of sclerostin expression and their effects are introduced and discussed herein based on recent and previous findings, including our research.

Angiotensin II Induces Aortic Rupture and Dissection in Osteoprotegerin-Deficient Mice.

Background The biological mechanism of action for osteoprotegerin, a soluble decoy receptor for the receptor activator of nuclear factor-kappa B ligand in the vascular structure, has not been elucidated. The study aim was to determine if osteoprotegerin affects aortic structural integrity in angiotensin II (Ang II)-induced hypertension. Methods and Results Mortality was higher (P<0.0001 by log-rank test) in 8-week-old male homozygotes of osteoprotegerin gene-knockout mice given subcutaneous administration of Ang II for 28 days, with an incidence of 21% fatal aortic rupture and 23% aortic dissection, than in age-matched wild-type mice. Ang II-infused aorta of wild-type mice showed that osteoprotegerin immunoreactivity was present with proteoglycan. The absence of osteoprotegerin was associated with decreased medial and adventitial thickness and increased numbers of elastin breaks as well as with increased periostin expression and soluble receptor activator of nuclear factor-kappa B ligand concentrations. PEGylated human recombinant osteoprotegerin administration decreased all-cause mortality (P<0.001 by log-rank test), the incidence of fatal aortic rupture (P=0.08), and aortic dissection (P<0.001) with decreasing numbers of elastin breaks, periostin expressions, and soluble receptor activator of nuclear factor-kappa B ligand concentrations in Ang II-infused osteoprotegerin gene-knockout mice. Conclusions These data suggest that osteoprotegerin protects against aortic rupture and dissection in Ang II-induced hypertension by inhibiting receptor activator of nuclear factor-kappa B ligand activity and periostin expression.

Evidence for the major contribution of remodeling-based bone formation in sclerostin-deficient mice.

Bone formation by osteoblasts is achieved through remodeling-based bone formation (RBBF) and modeling-based bone formation (MBBF). The former is when bone formation occurs after osteoclastic bone resorption to maintain bone mass and calcium homeostasis. The latter is when new bone matrices are added on the quiescent bone surfaces. Administration of anti-sclerostin neutralizing antibody promotes MBBF in ovariectomized rats and postmenopausal women. However, it remains to be elucidated which mode of bone formation mainly occurs in Sost-deficient mice under physiological conditions. Here, we show that two-thirds of bone formation involves RBBF in 12-week-old Sost-deficient mice (C57BL/6 background). Micro-computed tomography and histomorphometric analyses showed that the trabecular bone mass in Sost-KO mice was higher than that in Sost+/- mice. In contrast, the osteoclast number remained unchanged in Sost-KO mice, but the bone resorption marker TRAP5b in serum was slightly higher in those mice. Treatment with anti-RANKL antibody increased the trabecular bone mass of Sost+/- or Sost-KO mice. Bone formation markers such as osteoid surfaces, the mineral apposition rate, and bone formation rate were almost completely suppressed in Sost+/- mice treated with anti-RANKL antibody compared with vehicle-treated Sost+/- mice. In Sost-KO mice, treatment with anti-RANKL antibody suppressed those parameters by more than half. These findings indicate that RBBF accounts for most of the bone formation in Sost+/- mice, whereas approximately two-thirds of bone formation is estimated to be remodeling-based in 12-week-old Sost-deficient mice. Furthermore, anti-RANKL antibody may be useful for detecting MBBF on trabecular bone.

Inhibitor of protein kinase N3 suppresses excessive bone resorption in ovariectomized mice.

INTRODUCTION: The long-term inhibition of bone resorption suppresses new bone formation because these processes are coupled during physiological bone remodeling. The development of anti-bone-resorbing agents that do not suppress bone formation is urgently needed. We previously demonstrated that Wnt5a-Ror2 signaling in mature osteoclasts promoted bone-resorbing activity through protein kinase N3 (Pkn3). The p38 MAPK inhibitor SB202190 reportedly inhibited Pkn3 with a low Ki value (0.004 µM). We herein examined the effects of SB202190 on osteoclast differentiation and function in vitro and in vivo. MATERIALS AND METHODS: Bone marrow cells were cultured in the presence of M-csf and GST-Rankl to differentiate into multinucleated osteoclasts. Osteoclasts were treated with increasing concentrations of SB202190. For in vivo study, 10-week-old female mice were subjected to ovariectomy (OVX). OVX mice were intraperitoneally administered with a Pkn3 inhibitor at 2 mg/kg or vehicle for 4 weeks, and bone mass was analyzed by micro-CT. RESULTS: SB202190 suppressed the auto-phosphorylation of Pkn3 in osteoclast cultures. SB202190 significantly inhibited the formation of resorption pits in osteoclast cultures by suppressing actin ring formation. SB202190 reduced c-Src activity in osteoclast cultures without affecting the interaction between Pkn3 and c-Src. A treatment with SB202190 attenuated OVX-induced bone loss without affecting the number of osteoclasts or bone formation by osteoblasts. CONCLUSIONS: Our results showed that Pkn3 has potential as a therapeutic target for bone loss due to increased bone resorption. SB202190 is promising as a lead compound for the development of novel anti-bone-resorbing agents.

Characterization, pharmacokinetics, and pharmacodynamics of anti-Siglec-15 antibody and its potency for treating osteoporosis and as follow-up treatment after parathyroid hormone use.

Recent studies have established the idea that Siglec-15 is involved in osteoclast differentiation and/or function, and it is anticipated that therapies suppressing Siglec-15 function can be used to treat bone diseases such as osteoporosis. We have produced rat monoclonal anti-Siglec-15 antibody (32A1) and successively generated humanized monoclonal anti-Siglec-15 antibody (DS-1501a) from 32A1. Studies on the biological properties of DS-1501a showed its specific binding affinity to Siglec-15 and strong activity to inhibit osteoclastogenesis. 32A1 inhibited multinucleation of osteoclasts and bone resorption (pit formation) in cultured mouse bone marrow cells. 32A1 also inhibited pit formation in cultured human osteoclast precursor cells. Maximum serum concentration and serum exposure of DS-1501a in rats were increased in a dose-dependent manner after single subcutaneous or intravenous administration. Furthermore, single administration of DS-1501a significantly suppressed bone resorption markers with minimal effects on bone formation markers and suppressed the decrease in bone mineral density (BMD) of the lumbar vertebrae in ovariectomized (OVX) rats. In histological analysis, the osteoclasts distant from the chondro-osseous junction of the tibia tended to be flattened, shrunken, and functionally impaired in 32A1-treated rats, while alkaline phosphatase-positive osteoblasts were observed throughout the metaphyseal trabeculae. In addition, we compared the efficacy of 32A1 with that of alendronate (ALN) as follow-up medicine after treatment with parathyroid hormone (PTH) using mature established osteoporosis rats. The beneficial effect of PTH on bone turnover disappeared 8 weeks after discontinuing the treatment. The administration of 32A1 once every 4 weeks for 8 weeks suppressed bone resorption and bone formation when the treatment was switched from PTH to 32A1, leading to the maintenance of BMD and bone strength. Unlike with ALN, the onset of suppression of bone resorption with 32A1 was rapid, while the suppression of bone formation was mild. The improvement of bone mass, beneficial bone turnover balance, and suppression of osteoclast differentiation/multinucleation achieved by 32A1 were supported by histomorphometry. Notably, the effects of 32A1 on bone strength, not only structural (extrinsic) but also material (intrinsic) properties, were significantly greater than those of ALN. Since the effect of 32A1 on BMD was moderate, its effect on bone strength could not be fully explained by the increase in BMD. The beneficial balance of bone turnover caused by 32A1 might, at least in part, be responsible for the improvement in bone quality. This is the first report describing the effects of anti-Siglec-15 antibody in OVX rats; the findings suggest that this antibody could be an excellent candidate for treating osteoporosis, especially in continuation therapy after PTH treatment, due to its rapid action and unprecedented beneficial effects on bone quality.

Effector memory CD4+T cells in mesenteric lymph nodes mediate bone loss in food-allergic enteropathy model mice, creating IL-4 dominance.

Intestinal inflammation can be accompanied by osteoporosis, but their relationship, mediated by immune responses, remains unclear. Here, we investigated a non-IgE-mediated food-allergic enteropathy model of ovalbumin (OVA) 23-3 mice expressing OVA-specific T-cell-receptor transgenes. Mesenteric lymph nodes (MLNs) and their pathogenic CD4+T cells were important to enteropathy occurrence and exacerbation when the mice were fed an egg-white (EW) diet. EW-fed OVA23-3 mice also developed bone loss and increased CD44hiCD62LloCD4+T cells in the MLNs and bone marrow (BM); these changes were attenuated by MLN, but not spleen, resection. We fed an EW diet to F1 cross offspring from OVA23-3 mice and a mouse line expressing the photoconvertible protein KikGR to track MLN CD4+T cells. Photoconverted MLN CD44hiCD62LloCD4+T cells migrated predominantly to the BM; pit formation assay proved their ability to promote bone damage via osteoclasts. Significantly greater expression of IL-4 mRNA in MLN CD44hiCD62LloCD4+T cells and bone was observed in EW-fed OVA23-3 mice. Anti-IL-4 monoclonal antibody injection canceled bone loss in the primary inflammation phase in EW-fed mice, but less so in the chronic phase. This novel report shows the specific inflammatory relationship, via Th2-dominant-OVA-specific T cells and IL-4 production, between MLNs and bone, a distant organ, in food-allergic enteropathy.

Osteoclast differentiation by RANKL and OPG signaling pathways.

INTRODUCTION: In bone tissue, bone resorption by osteoclasts and bone formation by osteoblasts are repeated continuously. Osteoclasts are multinucleated cells that derive from monocyte-/macrophage-lineage cells and resorb bone. In contrast, osteoblasts mediate osteoclastogenesis by expressing receptor activator of nuclear factor-kappa B ligand (RANKL), which is expressed as a membrane-associated cytokine. Osteoprotegerin (OPG) is a soluble RANKL decoy receptor that is predominantly produced by osteoblasts and which prevents osteoclast formation and osteoclastic bone resorption by inhibiting the RANKL-RANKL receptor interaction. MATERIALS AND METHODS: In this review, we would like to summarize our experimental results on signal transduction that regulates the expression of RANKL and OPG. RESULTS: Using OPG gene-deficient mice, we have demonstrated that OPG and sclerostin produced by osteocytes play an important role in the maintenance of cortical and alveolar bone. In addition, it was shown that osteoclast-derived leukemia inhibitory factor (LIF) reduces the expression of sclerostin in osteocytes and promotes bone formation. WP9QY (W9) is a peptide that was designed to be structurally similar to one of the cysteine-rich TNF-receptortype-I domains. Addition of the W9 peptide to bone marrow culture simultaneously inhibited osteoclast differentiation and stimulated osteoblastic cell proliferation. An anti-sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) antibody inhibited multinucleated osteoclast formation induced by RANKL and macrophage colony-stimulating factor (M-CSF). Pit-forming activity of osteoclasts was also inhibited by the anti-Siglec-15 antibody. In addition, anti-Siglec-15 antibody treatment stimulated the appearance of osteoblasts in cultures of mouse bone marrow cells in the presence of RANKL and M-CSF. CONCLUSIONS: Bone mass loss depends on the RANK-RANKL-OPG system, which is a major regulatory system of osteoclast differentiation induction, activation, and survival.

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.

Sclerostin expression in trabecular bone is downregulated by osteoclasts.

Bone tissues have trabecular bone with a high bone turnover and cortical bone with a low turnover. The mechanisms by which the turnover rate of these bone tissues is determined remain unclear. Osteocytes secrete sclerostin, a Wnt/ß-catenin signaling antagonist, and inhibit bone formation. We found that sclerostin expression in cortical bone is more marked than in trabecular bone in Sost reporter mice. Leukemia inhibitory factor (LIF) secreted from osteoclasts reportedly suppressed sclerostin expression and promoted bone formation. Here, we report that osteoclasts downregulate sclerostin expression in trabecular bone and promote bone turnover. Treatment of C57BL/6 mice with an anti-RANKL antibody eliminated the number of osteoclasts and LIF-positive cells in trabecular bone. The number of sclerostin-positive cells was increased in trabecular bone, while the number of ß-catenin-positive cells and bone formation were decreased in trabecular bone. Besides, Tnfsf11 heterozygous (Rankl+/-) mice exhibited a decreased number of LIF-positive cells and increased number of sclerostin-positive cells in trabecular bone. Rankl+/- mice exhibited a decreased number of ß-catenin-positive cells and reduced bone formation in trabecular bone. Furthermore, in cultured osteoclasts, RANKL stimulation increased Lif mRNA expression, suggesting that RANKL signal increased LIF expression. In conclusion, osteoclasts downregulate sclerostin expression and promote trabecular bone turnover.

The Regulation of Bone Metabolism and Disorders by Wnt Signaling.

Wnt, a secreted glycoprotein, has an approximate molecular weight of 40 kDa, and it is a cytokine involved in various biological phenomena including ontogeny, morphogenesis, carcinogenesis, and maintenance of stem cells. The Wnt signaling pathway can be classified into two main pathways: canonical and non-canonical. Of these, the canonical Wnt signaling pathway promotes osteogenesis. Sclerostin produced by osteocytes is an inhibitor of this pathway, thereby inhibiting osteogenesis. Recently, osteoporosis treatment using an anti-sclerostin therapy has been introduced. In this review, the basics of Wnt signaling, its role in bone metabolism and its involvement in skeletal disorders have been covered. Furthermore, the clinical significance and future scopes of Wnt signaling in osteoporosis, osteoarthritis, rheumatoid arthritis and neoplasia are discussed.

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.

Blockade of the angiotensin II type 1 receptor increases bone mineral density and left ventricular contractility in a mouse model of juvenile Paget disease.

Juvenile Paget disease (JPD1), an autosomal-recessive disorder, is characterized by extremely rapid bone turnover due to osteoprotegerin deficiency. Its extra-skeletal manifestations, such as hypertension and heart failure, suggest a pathogenesis with shared skeletal and cardiovascular system components. In spite of this, the effects of anti-hypertensive drugs on bone morphometry remain unknown. We administered an angiotensin II type 1 receptor blocker, olmesartan (5 mg/kg/day) to 8-week-old male mice lacking the osteoprotegerin gene, with and without 1 µg/kg/min of angiotensin II infusion for 14 days. Olmesartan treatment decreased systolic blood pressure, and echocardiography showed increased left ventricular systolic contractility. Three-dimensional micro-computed tomography scans demonstrated that olmesartan treatment increased trabecular bone volume (sham, +176%; angiotensin II infusion, +335%), mineral density (sham, +150%; angiotensin II infusion, +313%), and trabecular number (sham, +407%; angiotensin II infusion, +622%) in the tibia. Olmesartan increased cortical mineral density (sham, +19%; angiotensin II infusion, +24%), decreased the cortical bone section area (sham, -16%; angiotensin II infusion, -18%), decreased thickness (sham, -18%; angiotensin II infusion, -31%), and decreased the lacunar area (sham, -41%; angiotensin II infusion, -27%) in the tibia. Similar trend was observed in the femur. Moreover, olmesartan decreased angiotensin II-induced increases in tartrate-resistant acid phosphatase concentrations in plasma, but it affected neither type I procollagen N-terminal propeptides, nor the receptor activator of nuclear factor kappa-B ligand. Our data suggest that blockade of the angiotensin II type 1 receptor improves bone vulnerability, and helps to maintain the heart's structural integrity in osteoprotegerin-deficient mice.

[Regulations of osteoclast formation and function by Wnt signals.]

Wnt ligands activate ß-catenin-dependent canonical and -independent non-canonical signaling pathways. Recent studies established importance of Wnt/ß-catenin signaling in bone accrual. Antibodies against the Wnt inhibitor sclerostin and those against the Wnt inhibitor Dickkopf-1 have been shown to be effective for increasing bone mass. In addition to their effects on bone formation, roles of Wnt signals in bone resorption are gradually clarified. In this review, we would like to introduce recent advances in roles of Wnt signals in osteoclast formation and functions and regulation of sclerostin expression by osteoclasts.

Regulatory mechanisms of sclerostin expression during bone remodeling.

Osteocytes are embedded in bone matrices and are connected to each other to respond to mechanical loading on bone. Recent studies have demonstrated the roles of mechanical loading in bone accrual. Bone responds to mechanical loading by decreasing the expression of sclerostin, an inhibitor of Wnt/ß-catenin signals, in osteocytes. This increases bone mass because the activation of Wnt/ß-catenin signals in bone microenvironments promotes bone formation and suppresses bone resorption. Thus, in recent years, sclerostin have attracted increasing attention in bone metabolism. However, the regulatory mechanism of sclerostin expression during bone remodeling has not been fully elucidated. In this review, we summarized the regulation of bone formation and resorption by Wnt signals, a Wnt/ß-catenin signal inhibitor sclerostin, and molecular mechanisms by which the expression of sclerostin is suppressed by mechanical loading and parathyroid hormone. We also discuss a possibility that osteoclasts suppress the expression of sclerostin during bone remodeling, which in turn, promote bone formation. The effectiveness of an anti-sclerostin antibody with anti-dickkopf-1 antibody for increasing bone mass was discussed.

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.

A Jak1/2 inhibitor, baricitinib, inhibits osteoclastogenesis by suppressing RANKL expression in osteoblasts in vitro.

The Janus kinases (Jaks) are hubs in the signaling process of more than 50 cytokine or hormone receptors. However, the function of Jak in bone metabolism remains to be elucidated. Here, we showed that the inhibition of Jak1 and/or Jak2 in osteoblast-lineage cells led to impaired osteoclastogenesis due to the reduced expression of receptor activator of nuclear factor-κB ligand (RANKL). Murine calvaria-derived osteoblasts induced differentiation of bone marrow cells into osteoclasts in the presence of 1,25-dihydroxyvitamin D3 (1,25D3) and prostaglandin E2 (PGE2) in vitro. However, treatment with the Jak1/2 inhibitor, baricitinib, markedly inhibited osteoclastogenesis in the co-culture. On the other hand, baricitinib did not inhibit RANKL-induced osteoclast differentiation of bone marrow macrophages. These results indicated that baricitinib acted on osteoblasts, but not on bone marrow macrophages. Baricitinib suppressed 1,25D3 and PGE2-induced up-regulation of RANKL in osteoblasts, but not macrophage colony-stimulating factor expression. Moreover, the addition of recombinant RANKL to co-cultures completely rescued baricitinib-induced impairment of osteoclastogenesis. shRNA-mediated knockdown of Jak1 or Jak2 also suppressed RANKL expression in osteoblasts and inhibited osteoclastogenesis. Finally, cytokine array revealed that 1,25D3 and PGE2 stimulated secretion of interleukin-6 (IL-6), IL-11, and leukemia inhibitory factor in the co-culture. Hence, Jak1 and Jak2 represent novel therapeutic targets for osteoporosis as well as inflammatory bone diseases including rheumatoid arthritis.

Bone Formation Is Coupled to Resorption Via Suppression of Sclerostin Expression by Osteoclasts.

Bone formation is coupled to bone resorption throughout life. However, the coupling mechanisms are not fully elucidated. Using Tnfrsf11b-deficient (OPG-/- ) mice, in which bone formation is clearly coupled to bone resorption, we found here that osteoclasts suppress the expression of sclerostin, a Wnt antagonist, thereby promoting bone formation. Wnt/ß-catenin signals were higher in OPG-/- and RANKL-transgenic mice with a low level of sclerostin. Conditioned medium from osteoclast cultures (Ocl-CM) suppressed sclerostin expression in UMR106 cells and osteocyte cultures. In vitro experiments revealed that osteoclasts secreted leukemia inhibitory factor (LIF) and inhibited sclerostin expression. Anti-RANKL antibodies, antiresorptive agents, suppressed LIF expression and increased sclerostin expression, thereby reducing bone formation in OPG-/- mice. Taken together, osteoclast-derived LIF regulates bone turnover through sclerostin expression. Thus, LIF represents a target for improving the prolonged suppression of bone turnover by antiresorptive agents. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

[Pharmacokinetics of bisphosphonate.]

The objective of treating osteoporosis is to prevent fractures. Bisphosphonates inhibit bone resorption and increase bone density, thereby suppressing the occurrence of fractures. Bisphosphonates have a high affinity for hydroxyapatite. About 20-80%of bisphosphonates absorbed in vivo is adsorbed onto the bone surface. However, the absorption rates of orally ingested bisphosphonates are less than 1%. Bisphosphonates adhered to the bone surface are specifically incorporated into cells upon bone resorption by osteoclasts. Thus, the cytoskeleton of osteoclasts is destroyed, and apoptosis is induced to suppress bone resorption. This article overviews the mechanisms of action and pharmacokinetics of bisphosphonates.