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.

Tyrosine kinases Btk and Tec regulate osteoclast differentiation by linking RANK and ITAM signals.

Certain autoimmune diseases result in abnormal bone homeostasis, but association of immunodeficiency with bone is poorly understood. Osteoclasts, which derive from bone marrow cells, are under the control of the immune system. Differentiation of osteoclasts is mainly regulated by signaling pathways activated by RANK and immune receptors linked to ITAM-harboring adaptors. However, it is unclear how the two signals merge to cooperate in osteoclast differentiation. Here we report that mice lacking the tyrosine kinases Btk and Tec show severe osteopetrosis caused by a defect in bone resorption. RANK and ITAM signaling results in formation of a Btk(Tec)/BLNK(SLP-76)-containing complex and PLCgamma-mediated activation of an essential calcium signal. Furthermore, Tec kinase inhibition reduces osteoclastic bone resorption in models of osteoporosis and inflammation-induced bone destruction. Thus, this study reveals the importance of the osteoclastogenic signaling complex composed of tyrosine kinases, which may provide the molecular basis for a new therapeutic strategy.

Induction of chondrogenesis with a RANKL-binding peptide, WP9QY, in vitro and in vivo in a rabbit model.

WP9QY (W9) is a receptor activator of nuclear factor-κB ligand (RANKL)-binding peptide that inhibits osteoclastogenesis by blunting the RANKL-RANK interaction, and also increases osteoblastogenesis via RANKL reverse signaling. W9 has dual effects on osteoclasts and osteoblasts; however, it is unknown whether the peptide has an effect on chondrocytes. Here, we report that W9 induces proliferation and differentiation of chondrocytes in vitro and repairs full-thickness articular cartilage defects in vivo. W9 stimulated chondrocyte differentiation in a two-dimensional (2D) culture of human mesenchymal stem cells (hMSCs), and transforming growth factor ß3 (TGF-ß3) showed synergistic effects with W9 on chondrogenesis. W9 enlarged the size of 3D pellet cultures of hMSCs and produced chondrocyte-specific matrices, especially in combined treatment with TGF-ß3. The peptide also stimulated proliferation of hMSCs with induction of expression of chondrogenesis-related genes. Several RANKL inhibitors had no effect on chondrocytic differentiation. RANKL-knockdown experiments showed that W9 did not induce chondrogenesis through RANKL, but did induce osteoblastogenesis through RANKL. Intraarticular injection of W9 resulted in significant repair of full-thickness articular cartilage defects in rabbits. Taken together, these results suggest that W9 ameliorates the articular cartilage defects by increasing the volume of cartilaginous matrices with accompanying induction of proliferation and differentiation of chondrocytes via mechanisms independent of RANKL inhibition and RANKL reverse signaling. Since no pharmaceuticals are clinically available for treatment of cartilage damage such as osteoarthritis, our findings demonstrate the potential of W9 to address the unmet medical needs.

Development of a conventional immunochemical detection system for determination of Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine in methylglyoxal-modified proteins.

Methylglyoxal (MGO) produced during glycolysis is known to react with arginine residues on proteins to generate advanced glycation end products, such as Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine (MG-H1). Since the production of MGO is increased during hyperglycemia or metabolic disorders in vivo, it is considered that the measurement of MG-H1 is useful for evaluating abnormalities in carbohydrate metabolism. Thus, we prepared a monoclonal antibody against MG-H1 to develop a conventional measurement system for MG-H1. Reactivity and specificity of the antibody to MGO-modified protein were confirmed by enzyme-linked immunosorbent assay and western blotting, respectively. The measurement of MG-H1 content by the antibody was positively correlated with that by electrospray ionization-liquid chromatography-tandem mass spectrometry and the ratio of modified arginine residues by amino acid analysis. Our results demonstrated that immunochemical methods could be useful for the estimation of MG-H1 content in modified proteins.

Discovery of the RANKL/RANK/OPG system.

Almost a quarter century has passed since discovery of receptor activator of NF-κB ligand (RANKL). This discovery had a major impact on identification of mechanisms regulating osteoclast differentiation and function, establishment of a research field bridging bone and the immune system (osteoimmunology), and development of a fully human anti-RANKL neutralizing antibody (denosumab). Denosumab is now clinically available for treatment of osteoporosis and cancer-induced bone diseases in the US, Europe and many other countries, including Japan. Denosumab is a so-called blockbuster drug, with sales of 5.0 billion US dollars in 2019. This is a real success story from bench to bedside. In this review, the pivotal roles of the RANKL/RANK/OPG system in osteoclast differentiation and function are shown. RANKL is a ligand required for osteoclast generation, RANK is the receptor for RANKL, and osteoprotegerin (OPG) is a decoy receptor for RANKL. The review covers recent results showing the importance of RANKL on osteoblasts in regulation of osteogenesis and the role of RANKL-RANK dual signaling in coupling of bone resorption and formation, including demonstration of RANKL reverse signaling that we had previously hypothesized. Possible applications of anti-RANKL antibody in treatment of cancer are also discussed.

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.

Kif1c regulates osteoclastic bone resorption as a downstream molecule of p130Cas.

Podosome formation in osteoclasts is an important initial step in osteoclastic bone resorption. Mice lacking c-Src (c-Src-/- ) exhibited osteopetrosis due to a lack of podosome formation in osteoclasts. We previously identified p130Cas (Crk-associated substrate [Cas]) as one of c-Src downstream molecule and osteoclast-specific p130Cas-deficient (p130CasΔOCL-/- ) mice also exhibited a similar phenotype to c-Src-/- mice, indicating that the c-Src/p130Cas plays an important role for bone resorption by osteoclasts. In this study, we performed a cDNA microarray and compared the gene profiles of osteoclasts from c-Src-/- or p130CasΔOCL-/- mice with wild-type (WT) osteoclasts to identify downstream molecules of c-Src/p130Cas involved in bone resorption. Among several genes that were commonly downregulated in both c-Src-/- and p130CasΔOCL-/- osteoclasts, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization. Reduced Kif1c expression was observed in both c-Src-/- and p130CasΔOCL-/- osteoclasts compared with WT osteoclasts. Kif1c exhibited a broad tissue distribution, including osteoclasts. Knockdown of Kif1c expression using shRNAs in WT osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in p130CasΔOCL-/- osteoclasts but not c-Src-/- osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas (191 words). SIGNIFICANCE OF THE STUDY: We previously showed that the c-Src/p130Cas (Cas) plays an important role for bone resorption by osteoclasts. In this study, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization, as a downstream molecule of c-Src/p130Cas axis, using cDNA microarray. Knockdown of Kif1c expression using shRNAs in wild-type osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in osteoclast-specific p130Cas-deficient (p130CasΔOCL-/- ) osteoclasts but not c-Src-/- osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas.

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).

Peptide drugs accelerate BMP-2-induced calvarial bone regeneration and stimulate osteoblast differentiation through mTORC1 signaling.

Both W9 and OP3-4 were known to bind the receptor activator of NF-κB ligand (RANKL), inhibiting osteoclastogenesis. Recently, both peptides were shown to stimulate osteoblast differentiation; however, the mechanism underlying the activity of these peptides remains to be clarified. A primary osteoblast culture showed that rapamycin, an mTORC1 inhibitor, which was recently demonstrated to be an important serine/threonine kinase for bone formation, inhibited the peptide-induced alkaline phosphatase activity. Furthermore, both peptides promoted the phosphorylation of Akt and S6K1, an upstream molecule of mTORC1 and the effector molecule of mTORC1, respectively. In the in vivo calvarial defect model, W9 and OP3-4 accelerated BMP-2-induced bone formation to a similar extent, which was confirmed by histomorphometric analyses using fluorescence images of undecalcified sections. Our data suggest that these RANKL-binding peptides could stimulate the mTORC1 activity, which might play a role in the acceleration of BMP-2-induced bone regeneration by the RANKL-binding peptides.

Osteoblast-derived Laminin-332 is a novel negative regulator of osteoclastogenesis in bone microenvironments.

Laminin-332 (Lm-332), a major basement membrane protein, has been shown to provide a niche for some stem cells. Here, we found that Lm-332 was expressed in osteoblasts, and is implicated in the regulation of osteoclast differentiation. Immunofluorescence analysis of laminin-ß3, a unique component of Lm-332, indicated specific expression of laminin-ß3 in osteoblast-like cells localized on bone surface. RT-PCR analysis confirmed that α3, ß3, and γ2 chains of Lm-332 were all expressed in primary osteoblasts prepared from mouse calvaria. Lm-332 markedly inhibited osteoclastogenesis induced by receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) when bone marrow-derived macrophages (BMMs) were cultured on Lm-332-coated plates. Lm-332 also blocked RANKL-induced activation of mitogen-activated protein kinases (MAPKs) (ERK, JNK, and p38) and expression of NFATc1, c-Fos, and c-Jun. Lm-332 suppressed osteoclast differentiation while retaining macrophage phenotypes, including nonspecific esterase activity and gene expression of lysozyme and EGF-like module-containing mucin-like hormone receptor-like 1 (Emr1). Furthermore, the treatment of primary osteoblasts with osteoclastogenic factors dramatically suppressed expression of Lm-332. These findings suggest that Lm-332 produced by osteoblasts in bone tissues has a pivotal role in controlling normal bone remodeling through suppressing osteoclastogenesis.

The thymic cortical epithelium determines the TCR repertoire of IL-17-producing γδT cells.

The thymus provides a specialized microenvironment in which distinct subsets of thymic epithelial cells (TECs) support T-cell development. Here, we describe the significance of cortical TECs (cTECs) in T-cell development, using a newly established mouse model of cTEC deficiency. The deficiency of mature cTECs caused a massive loss of thymic cellularity and impaired the development of αßT cells and invariant natural killer T cells. Unexpectedly, the differentiation of certain γδT-cell subpopulations-interleukin-17-producing Vγ4 and Vγ6 cells-was strongly dysregulated, resulting in the perturbation of γδT-mediated inflammatory responses in peripheral tissues. These findings show that cTECs contribute to the shaping of the TCR repertoire, not only of "conventional" αßT cells but also of inflammatory "innate" γδT cells.

Establishment of a xenograft model to explore the mechanism of bone destruction by human oral cancers and its application to analysis of role of RANKL.

BACKGROUND: The molecular mechanism underlying bone invasion caused by oral squamous cell carcinoma (OSCC) is not well understood. To elucidate the molecular mechanism, the development of more suitable xenograft models mimicking human mandibular bone destruction by OSCC has been required. MATERIALS AND METHODS: Human OSCC cell lines, HSC3, HSC3-C1, and HSC3-R2, were injected in the periosteal region of the mandible in athymic mice, and the bone destruction was analyzed. Receptor activators of nuclear factor κ-B ligand (RANKL) mRNA and protein expression levels were measured in the OSCC cell lines. Antibody that specifically neutralizes mouse RANKL and human RANKL, respectively, was injected into HSC3-cell-transplanted mice. RESULTS: Transplantation of HSC3 cells induced mandibular bone destruction. Histological examination revealed numerous osteoclasts on the bone destruction surface. Fibroblastic cell intervention between the cancer nests and resorbing bone surface was observed in a similar fashion to those observed in human OSCC cases. The number of osteoclasts and fibroblasts was significantly correlated. Bone destruction induced by the transplantation of HSC3 cells was reduced by injection of an antibody that specifically neutralizes mouse RANKL. Transplantation of HSC3-R2 cells, which overexpresses RANKL, induced advanced bone destruction compared to that of HSC3-C1 cells, which only overexpress the empty vector. CONCLUSIONS: We established a useful xenograft model for investigating the molecular mechanism underlying the bone destruction induced by OSCC in the jaw. This model will be used to investigate the precise roles of several cytokines synthesized by both cancer cells and fibroblastic cells in OSCC-associated bone destruction in the jaw.

Control of mammary stem cell function by steroid hormone signalling.

The ovarian hormones oestrogen and progesterone profoundly influence breast cancer risk, underpinning the benefit of endocrine therapies in the treatment of breast cancer. Modulation of their effects through ovarian ablation or chemoprevention strategies also significantly decreases breast cancer incidence. Conversely, there is an increased risk of breast cancer associated with pregnancy in the short term. The cellular mechanisms underlying these observations, however, are poorly defined. Here we demonstrate that mouse mammary stem cells (MaSCs) are highly responsive to steroid hormone signalling, despite lacking the oestrogen and progesterone receptors. Ovariectomy markedly diminished MaSC number and outgrowth potential in vivo, whereas MaSC activity increased in mice treated with oestrogen plus progesterone. Notably, even three weeks of treatment with the aromatase inhibitor letrozole was sufficient to reduce the MaSC pool. In contrast, pregnancy led to a transient 11-fold increase in MaSC numbers, probably mediated through paracrine signalling from RANK ligand. The augmented MaSC pool indicates a cellular basis for the short-term increase in breast cancer incidence that accompanies pregnancy. These findings further indicate that breast cancer chemoprevention may be achieved, in part, through suppression of MaSC function.

RelB-induced expression of Cot, an MAP3K family member, rescues RANKL-induced osteoclastogenesis in alymphoplasia mice by promoting NF-κB2 processing by IKKα.

The alternative nuclear factor-κB (NF-κB) pathway, mainly the RelB-p52 heterodimer, plays important roles in bone metabolism through an unknown mechanism. We have previously reported that alymphoplasia (aly/aly) mice, which lack active NF-κB-inducing kinase (NIK), show mild osteopetrosis due to the inhibition of osteoclastogenesis. p100 retains RelB in the cytoplasm and inhibits RANKL-induced osteoclastogenesis in aly/aly cells. Furthermore, the overexpression of RelB in aly/aly cells rescues RANKL-induced osteoclastogenesis by inducing p100 processing. In contrast, the overexpression of p65 in aly/aly cells has no effect. However, the overexpression of RelB fails to rescue RANKL-induced osteoclastogenesis in the presence of p100ΔGRR, which cannot be processed to p52, suggesting that p100 processing is a key step in RelB-rescued, RANKL-induced osteoclastogenesis in aly/aly cells. In this study, Cot (cancer Osaka thyroid), an MAP3K, was up-regulated by RelB overexpression. Analysis of the Cot promoter demonstrated that p65 and RelB bound to the distal NF-κB-binding site and that RelB but not p65 bound to the proximal NF-κB-binding site in the Cot promoter. The knocking down of Cot expression significantly reduced the RANKL-induced osteoclastogenesis induced by RelB overexpression. The phosphorylation of IKKα at threonine 23 and its kinase activity were indispensable for the processing of p100 and osteoclastogenesis by RelB-induced Cot. Finally, constitutively activated Akt enhanced osteoclastogenesis by RelB-induced Cot, and a dominant-negative form of Akt significantly inhibited it. Taken together, these results indicate that the overexpression of RelB restores RANKL-induced osteoclastogenesis by activation of Akt/Cot/IKKα-induced p100 processing.

Porphyromonas gingivalis-derived lysine gingipain enhances osteoclast differentiation induced by tumor necrosis factor-α and interleukin-1ß but suppresses that by interleukin-17A: importance of proteolytic degradation of osteoprotegerin by lysine gingipain.

Periodontitis is a chronic inflammatory disease accompanied by alveolar bone resorption by osteoclasts. Porphyromonas gingivalis, an etiological agent for periodontitis, produces cysteine proteases called gingipains, which are classified based on their cleavage site specificity (i.e. arginine (Rgps) and lysine (Kgps) gingipains). We previously reported that Kgp degraded osteoprotegerin (OPG), an osteoclastogenesis inhibitory factor secreted by osteoblasts, and enhanced osteoclastogenesis induced by various Toll-like receptor (TLR) ligands (Yasuhara, R., Miyamoto, Y., Takami, M., Imamura, T., Potempa, J., Yoshimura, K., and Kamijo, R. (2009) Lysine-specific gingipain promotes lipopolysaccharide- and active-vitamin D3-induced osteoclast differentiation by degrading osteoprotegerin. Biochem. J. 419, 159-166). Osteoclastogenesis is induced not only by TLR ligands but also by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-17A, in inflammatory conditions, such as periodontitis. Although Kgp augmented osteoclastogenesis induced by TNF-α and IL-1ß in co-cultures of mouse osteoblasts and bone marrow cells, it suppressed that induced by IL-17A. In a comparison of proteolytic degradation of these cytokines by Kgp in a cell-free system with that of OPG, TNF-α and IL-1ß were less susceptible, whereas IL-17A and OPG were equally susceptible to degradation by Kgp. These results indicate that the enhancing effect of Kgp on cytokine-induced osteoclastogenesis is dependent on the difference in degradation efficiency between each cytokine and OPG. In addition, elucidation of the N-terminal amino acid sequences of OPG fragments revealed that Kgp primarily cleaved OPG in its death domain homologous region, which might prevent dimer formation of OPG required for inhibition of receptor activator of nuclear factor κB ligand. Collectively, our results suggest that degradation of OPG by Kgp is a crucial event in the development of osteoclastogenesis and bone loss in periodontitis.

Systemic circulation and bone recruitment of osteoclast precursors tracked by using fluorescent imaging techniques.

Osteoclasts are bone-resorbing polykaryons differentiated from monocyte/macrophage-lineage hematopoietic precursors. It remains unclear whether osteoclasts originate from circulating blood monocytes or from bone tissue-resident precursors. To address this question, we combined two different experimental procedures: 1) shared blood circulation "parabiosis" with fluorescently labeled osteoclast precursors, and 2) photoconversion-based cell tracking with a Kikume Green-Red protein (KikGR). In parabiosis, CX(3)CR1-EGFP knock-in mice in which osteoclast precursors were labeled with EGFP were surgically connected with wild-type mice to establish a shared circulation. Mature EGFP(+) osteoclasts were found in the bones of the wild-type mice, indicating the mobilization of EGFP(+) osteoclast precursors into bones from systemic circulation. Receptor activator for NF-κB ligand stimulation increased the number of EGFP(+) osteoclasts in wild-type mice, suggesting that this mobilization depends on the bone resorption state. Additionally, KikGR(+) monocytes (including osteoclast precursors) in the spleen were exposed to violet light, and 2 d later we detected photoconverted "red" KikGR(+) osteoclasts along the bone surfaces. These results indicate that circulating monocytes from the spleen entered the bone spaces and differentiated into mature osteoclasts during a certain period. The current study used fluorescence-based methods clearly to demonstrate that osteoclasts can be generated from circulating monocytes once they home to bone tissues.

Spleen serves as a reservoir of osteoclast precursors through vitamin D-induced IL-34 expression in osteopetrotic op/op mice.

Osteoclasts are generated from monocyte/macrophage-lineage precursors in response to colony-stimulating factor 1 (CSF-1) and receptor activator of nuclear factor-κB ligand (RANKL). CSF-1-mutated CSF-1(op/op) mice as well as RANKL(-/-) mice exhibit osteopetrosis (OP) caused by osteoclast deficiency. We previously identified RANKL receptor (RANK)/CSF-1 receptor (CSF-1R) double-positive cells as osteoclast precursors (OCPs), which existed in bone in RANKL(-/-) mice. Here we show that OCPs do not exist in bone but in spleen in CSF-1(op/op) mice, and spleen acts as their reservoir. IL-34, a newly discovered CSF-1R ligand, was highly expressed in vascular endothelial cells in spleen in CSF-1(op/op) mice. Vascular endothelial cells in bone also expressed IL-34, but its expression level was much lower than in spleen, suggesting a role of IL-34 in the splenic generation of OCPs. Splenectomy (SPX) blocked CSF-1-induced osteoclastogenesis in CSF-1(op/op) mice. Osteoclasts appeared in aged CSF-1(op/op) mice with up-regulation of IL-34 expression in spleen and bone. Splenectomy blocked the age-associated appearance of osteoclasts. The injection of 2-methylene-19-nor-(20S)-1α,25(OH)(2)D(3) (2MD), a potent analog of 1α,25-dihidroxyvitamin D(3), into CSF-1(op/op) mice induced both hypercalcemia and osteoclastogenesis. Administration of 2MD enhanced IL-34 expression not only in spleen but also in bone through a vitamin D receptor-mediated mechanism. Either splenectomy or siRNA-mediated knockdown of IL-34 suppressed 2MD-induced osteoclastogenesis. These results suggest that IL-34 plays a pivotal role in maintaining the splenic reservoir of OCPs, which are transferred to bone in response to diverse stimuli, in CSF-1(op/op) mice. The present study also suggests that the IL-34 gene in vascular endothelial cells is a unique target of vitamin D.

Stimulation of bone formation in cortical bone of mice treated with a receptor activator of nuclear factor-κB ligand (RANKL)-binding peptide that possesses osteoclastogenesis inhibitory activity.

To date, parathyroid hormone is the only clinically available bone anabolic drug. The major difficulty in the development of such drugs is the lack of clarification of the mechanisms regulating osteoblast differentiation and bone formation. Here, we report a peptide (W9) known to abrogate osteoclast differentiation in vivo via blocking receptor activator of nuclear factor-κB ligand (RANKL)-RANK signaling that we surprisingly found exhibits a bone anabolic effect in vivo. Subcutaneous administration of W9 three times/day for 5 days significantly augmented bone mineral density in mouse cortical bone. Histomorphometric analysis showed a decrease in osteoclastogenesis in the distal femoral metaphysis and a significant increase in bone formation in the femoral diaphysis. Our findings suggest that W9 exerts bone anabolic activity. To clarify the mechanisms involved in this activity, we investigated the effects of W9 on osteoblast differentiation/mineralization in MC3T3-E1 (E1) cells. W9 markedly increased alkaline phosphatase (a marker enzyme of osteoblasts) activity and mineralization as shown by alizarin red staining. Gene expression of several osteogenesis-related factors was increased in W9-treated E1 cells. Addition of W9 activated p38 MAPK and Smad1/5/8 in E1 cells, and W9 showed osteogenesis stimulatory activity synergistically with BMP-2 in vitro and ectopic bone formation. Knockdown of RANKL expression in E1 cells reduced the effect of W9. Furthermore, W9 showed a weak effect on RANKL-deficient osteoblasts in alkaline phosphatase assay. Taken together, our findings suggest that this peptide may be useful for the treatment of bone diseases, and W9 achieves its bone anabolic activity through RANKL on osteoblasts accompanied by production of several autocrine factors.

Bone resorption facilitates osteoblastic bone metastatic colonization by cooperation of insulin-like growth factor and hypoxia.

Bone metastasis is a multistep process that includes cancer cell dissemination, colonization, and metastatic growth. Furthermore, this process involves complex, reciprocal interactions between cancer cells and the bone microenvironment. Bone resorption is known to be involved in both osteolytic and osteoblastic bone metastasis. However, the precise roles of the bone resorption in the multistep process of osteoblastic bone metastasis remain unidentified. In this study, we show that bone resorption plays important roles in cancer cell colonization during the initial stage of osteoblastic bone metastasis. We applied bioluminescence/X-ray computed tomography multimodal imaging that allows us to spatiotemporally analyze metastasized cancer cells and bone status in osteoblastic bone metastasis models. We found that treatment with receptor activator of factor-κB ligand (RANKL) increased osteoblastic bone metastasis when given at the same time as intracardiac injection of cancer cells, but failed to increase metastasis when given 4 days after cancer cell injection, suggesting that RANKL-induced bone resorption facilitates growth of cancer cells colonized in the bone. We show that insulin-like growth factor-1 released from the bone during bone resorption and hypoxia-inducible factor activity in cancer cells cooperatively promoted survival and proliferation of cancer cells in bone marrow. These results suggest a mechanism that bone resorption and hypoxic stress in the bone microenvironment cooperatively play an important role in establishing osteoblastic metastasis.