RESUMEN
The vitamin D receptor (VDR) is expressed most abundantly in osteoblasts and osteocytes (osteoblastic cells) in bone tissues and regulates bone resorption and calcium (Ca) and phosphate (P) homeostasis in association with parathyroid hormone (PTH). We previously reported that near-physiological doses of vitamin D compounds suppressed bone resorption through VDR in osteoblastic cells. We also found that supra-physiological doses of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced bone resorption and hypercalcemia via VDR in osteoblastic cells. Here, we report that the latter, a proresorptive dose of 1,25(OH)2D3, causes soft tissue calcification through VDR in osteoblastic cells. High concentrations of vitamin D affect multiple organs and cause soft tissue calcification, with increases in bone resorption and serum Ca levels. Such a variety of symptoms is known as hypervitaminosis D, which is caused by not only high doses of vitamin D but also impaired vitamin D metabolism and diseases that produce 1,25(OH)2D3 ectopically. To clarify the biological process hierarchy in hypervitaminosis D, a proresorptive dose of 1,25(OH)2D3 was administered to wild-type mice in which bone resorption had been suppressed by neutralizing anti-receptor activator of NF-κB ligand (RANKL) antibody. 1,25(OH)2D3 upregulated the serum Ca x P product, concomitantly induced calcification of the aorta, lungs, and kidneys, and downregulated serum PTH levels in control IgG-pretreated wild-type mice. Pretreatment of wild-type mice with anti-RANKL antibody did not affect the down-regulation of PTH levels by 1,25(OH)2D3, but inhibited the increase of the serum Ca x P product and soft tissue calcification induced by 1,25(OH)2D3. Consistent with the effects of anti-RANKL antibody, VDR ablation in osteoblastic cells also did not affect the down-regulation of PTH levels by 1,25(OH)2D3, but suppressed the 1,25(OH)2D3-induced increase of the serum Ca x P product and calcification of soft tissues. Taken together with our previous results, these findings suggest that bone resorption induced by VDR signaling in osteoblastic cells is critical for the pathogenesis of hypervitaminosis D, but PTH is not involved in hypervitaminosis D.
Asunto(s)
Fenómenos Biológicos , Receptores de Calcitriol , Ratones , Animales , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Hormona Paratiroidea/metabolismo , Calcitriol/metabolismo , Vitamina D/farmacología , Vitamina D/metabolismo , Vitaminas/farmacologíaRESUMEN
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.
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Proteínas Adaptadoras Transductoras de Señales , Proteína Morfogenética Ósea 2 , Animales , Masculino , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Osteogénesis , Microtomografía por Rayos X , Proteína Morfogenética Ósea 2/metabolismoRESUMEN
Discovery of RANKL (receptor activator of NF-κB ligand) had an impact on identification of the mechanisms regulating osteoclast differentiation and function, resulting in establishment of research field bridging bone biology and immunology (osteoimmunology), and development of a human anti-RANKL monoclonal antibody (denosumab). Denosumab has been clinically available for treatment of osteoporosis and cancer-induced bone diseases in many countries. Denosumab is a so-called blockbuster of which sales amount was 5.3 billion US dollars in 2021. I will discuss the intense competition between Amgen Inc. and us regarding discovery of RANKL. One of the recent topics is the identification of RANKL reverse signaling with a RANKL-binding peptide, W9 known as a RANKL antagonist. The RANKL reverse signaling stimulates differentiation of osteoblasts and bone formation. The findings revealed the RANKL-RANK (a receptor of RANKL) dual signaling in coupling between bone resorption and bone formation. Interestingly, W9 also stimulates differentiation of chondrocytes and repairs defect of articular cartilage regardless of RANKL. Identification of the mechanisms will be useful for development of pharmaceuticals treating osteoarthritis. I also suggest possible applications of anti-RANKL antibody (anti-RANKL) to the treatment of cancer patients. RANKL has an important role in development of medullary thymic epithelial cells (mTECs) establishing self-tolerance. Anti-RANKL potentiates anticancer immune responses thorough regeneration of tumor-reactive T-cells by inhibiting mTEC development. I expect the synergy of anti-RANKL and immune checkpoint inhibitors such as anti-CTLA-4 antibody and anti-PD-1 antibody for immuno-oncology. Several clinical trials are currently in progress. It is likely that cancers will not be incurable diseases in the near future.
Asunto(s)
Resorción Ósea , Osteoporosis , Humanos , Denosumab/uso terapéutico , Osteoclastos , Ligando RANK , Resorción Ósea/tratamiento farmacológico , Osteoporosis/terapia , HuesosRESUMEN
The chemokine fractalkine (FKN) is produced by various cell types, including osteoblasts and endothelial cells in bone tissue, and signals through a sole receptor, CX3CR1, which is expressed on monocytes/macrophages, including osteoclast precursors (OCPs). However, the direct effects of FKN signaling on osteoclast lineage cells under homeostatic noninflammatory conditions remain unclear. Here, we report that FKN regulates mouse OCP survival and primes OCPs for subsequent osteoclast differentiation. Wild-type but not CX3CR1-deficient OCPs grown on immobilized FKN showed enhanced osteoclast formation following receptor activator of NF-κB ligand (RANKL) stimulation, with increased expression of osteoclast differentiation markers. Interestingly, the growth of OCPs on immobilized FKN increased the expression of Cx3cr1 and Tnfrsf11a (Rank) transcripts, but following RANKL stimulation, OCPs rapidly downregulated Cx3cr1 expression. Consistently, anti-FKN monoclonal antibody (mAb) treatment attenuated RANKL-induced osteoclast formation on immobilized FKN before, but not during, RANKL stimulation. CX3CR1 and RANK proteins were highly expressed on bone marrow-derived CD11bhigh CD115+ OCPs. Growth on immobilized FKN prior to RANKL stimulation also increased CD11bhigh CD115+ OCP number and their survival and differentiation potential. In a RANKL-based mouse model of bone loss, anti-FKN mAb pretreatment significantly inhibited RANKL-dependent bone loss. Thus, blocking the FKN-CX3CR1 axis could represent a therapeutic option in noninflammatory bone loss diseases. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
RESUMEN
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.
Asunto(s)
Resorción Ósea , Osteogénesis , Proteínas Adaptadoras Transductoras de Señales , Animales , Femenino , Glicoproteínas/metabolismo , Humanos , Hiperostosis , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteogénesis/fisiología , Ratas , Sindactilia , Microtomografía por Rayos XRESUMEN
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.
Asunto(s)
Cartílago Articular , Condrogénesis , Animales , Cartílago Articular/metabolismo , Diferenciación Celular , Células Cultivadas , Condrocitos/metabolismo , Osteoclastos/metabolismo , Osteogénesis , Péptidos/metabolismo , Péptidos/farmacología , Conejos , Factor de Crecimiento Transformador beta3/metabolismoRESUMEN
OBJECTIVES: Osteoclasts are induced by macrophage colony-stimulating factor-1 (CSF-1) and receptor activator of nuclear factor-κB (RANK) ligand (RANKL). Monocyte/macrophage lineages are thought to be osteoclast precursors; however, such cells have not been fully characterized owing to a lack of tools for their identification. Osteoclast precursors express colony-stimulating factor-1 receptor (CSF-1R) and RANK. However, the capacity of conventional methods using anti-RANK antibodies to detect RANK+ cells by flow cytometry is insufficient. Here, we developed a high-sensitivity method for detecting RANK+ cells using biotinylated recombinant glutathione S-transferase-RANKL (GST-RANKL-biotin). METHODS: We sorted sub-populations of mouse bone marrow (BM) or peripheral blood (PB) cells using GST-RANKL-biotin, anti-CSF1R, and anti-B220 antibodies and induced osteoclastogenesis in vitro. RESULTS: The frequency of the RANK+ population in BM detected by GST-RANKL-biotin was significantly higher than that detected by anti-RANK antibodies. Although RANK+ cells were detected in both the B220+ and B220- populations, the macrophage lineage was present only in B220-. Unexpectedly, a significantly higher number of osteoclasts was induced in RANK-CSF-1R+ cells than in RANK+CSF-1R+ cells contained in the B220- population. In contrast, the PB-derived B220-RANK+CSF-1R+ population contained a significantly higher frequency of osteoclast precursors than the B220-RANK-CSF-1R+ population. CONCLUSIONS: These results suggest that GST-RANKL-biotin is useful for the detection of RANK+ cells and that RANK and CSF-1R may be helpful indicators of osteoclast precursors in PB.
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Células de la Médula Ósea , FN-kappa B , Animales , Células de la Médula Ósea/metabolismo , Proteínas Portadoras , Glicoproteínas de Membrana/metabolismo , Ratones , FN-kappa B/metabolismo , Osteoclastos/metabolismo , Receptor Activador del Factor Nuclear kappa-B/genéticaRESUMEN
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.
Asunto(s)
Osteoprotegerina/metabolismo , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Animales , Humanos , Japón , Modelos Biológicos , Osteoclastos/metabolismo , Osteogénesis/fisiologíaRESUMEN
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.
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Diferenciación Celular , Osteoclastos/citología , Osteoclastos/metabolismo , Osteoprotegerina/metabolismo , Ligando RANK/metabolismo , Transducción de Señal , Animales , Humanos , OsteogénesisRESUMEN
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.
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Imidazoles/química , Oligopéptidos/química , Ornitina/análogos & derivados , Ornitina/química , Piruvaldehído/química , InmunoquímicaRESUMEN
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.
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Resorción Ósea/genética , Linaje de la Célula/genética , Osteoblastos/metabolismo , Receptores de Calcitriol/fisiología , Vitamina D/análogos & derivados , Animales , Resorción Ósea/metabolismo , Huesos/efectos de los fármacos , Huesos/metabolismo , Femenino , Factor-23 de Crecimiento de Fibroblastos , Hipercalcemia/genética , Hipercalcemia/metabolismo , Hipercalcemia/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Ratones Transgénicos , Obesidad/genética , Obesidad/metabolismo , Obesidad/patología , Osteoblastos/citología , Receptores de Calcitriol/genética , Vitamina D/farmacologíaRESUMEN
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.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Remodelación Ósea/fisiología , Hueso Esponjoso/metabolismo , Osteoclastos/metabolismo , Ligando RANK/genética , Animales , Anticuerpos/inmunología , Biomarcadores de Tumor/metabolismo , Densidad Ósea , Hueso Cortical/metabolismo , Factor Inhibidor de Leucemia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ligando RANK/inmunología , Regulación hacia Arriba/genética , Vía de Señalización Wnt/fisiologíaRESUMEN
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.
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Enfermedades Óseas Metabólicas , Resorción Ósea , Anciano , Animales , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/prevención & control , Diferenciación Celular , Humanos , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Osteoclastos/metabolismo , Proteínas Serina-Treonina Quinasas , Ligando RANK/metabolismo , Quinasa de Factor Nuclear kappa BRESUMEN
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.
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Resorción Ósea , Proteína Sustrato Asociada a CrK/metabolismo , Regulación de la Expresión Génica , Cinesinas/metabolismo , Osteoclastos/metabolismo , Actinas/metabolismo , Animales , Huesos/metabolismo , Proteína Tirosina Quinasa CSK/genética , Proteína Tirosina Quinasa CSK/metabolismo , Células HEK293 , Heterocigoto , Humanos , Factor Estimulante de Colonias de Macrófagos/metabolismo , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Fosforilación , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , Dedos de ZincRESUMEN
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).
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Hueso Esponjoso/metabolismo , Homeostasis , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Hueso Esponjoso/citología , Ratones , Ratones Noqueados , Osteoblastos/citología , Osteoclastos/citología , Ligando RANK/genética , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/genética , Receptor Activador del Factor Nuclear kappa-B/metabolismoRESUMEN
Discovery of RANKL (receptor activator of NF-κB ligand) gave a great impact on identification of the mechanisms regulating osteoclast differentiation and function, establishment of research field bridging bone and mineral research and immunology (osteoimmunology), and development of a fully human anti-RANKL monoclonal neutralizing antibody (denosumab). Denosumab has been clinically available for treatment of osteoporosis and cancer-induced bone diseases in the US, Europe and many countries including Japan. Denosumab is a so-called blockbuster of which sales amount was 3.9 billion US dollars in 2017. Because RANKL is the absolute factor for osteoclast differentiation, anti-RANKL antibody is very effective and its application is good news for many patients. Recent topics are the identification of importance of RANKL on osteoblasts in regulation of osteogenesis and the demonstration of RANKL-RANK (the receptor of RANKL) dual signaling in coupling between bone resorption and bone formation. RANKL reverse signaling that we had hypothesized was demonstrated at last. In this review I describe the mechanism of anti-RANKL antibody in the treatment of metabolic bone diseases including osteoporosis. I also suggest possible applications of anti-RANKL antibody to the treatment of cancer patients.
Asunto(s)
Resorción Ósea , Denosumab/farmacología , Osteoporosis/terapia , Ligando RANK/inmunología , Anticuerpos Monoclonales Humanizados , Humanos , Osteoclastos/citologíaRESUMEN
We examined the effect of teriparatide, and switching from teriparatide to anti-RANKL (receptor activator of nuclear factor κB ligand) monoclonal antibody, in ovariectomized mice. Twelve-week-old female C57BL/6 mice were ovariectomized or sham operated. Four weeks after surgery, ovariectomized mice were subjected to one of the following four treatments: phosphate-buffered saline (PBS) for 8weeks; teriparatide for 4weeks followed by PBS for 4weeks (PTH4W group); teriparatide for 8weeks (PTH8W group); or teriparatide for 4weeks followed by anti-RANKL antibody (single subcutaneous injection of 5mg/kg) (SWITCH group). Twelve weeks after the operation, bone mineral density was increased in PTH8W and SWITCH groups to broadly comparable levels, but these were significantly decreased in the PTH4W group after discontinuation of teriparatide. Histomorphometric analysis demonstrated that cancellous bone formation and resorption were profoundly suppressed in the SWITCH group. Bone formation was also suppressed on the endocortical surface of cortical bone but was maintained on the periosteal surface. Anti-RANKL antibody suppressed osteoclast activity immediately after treatment, while bone formation was only gradually decreased. These results suggest that anti-RANKL antibody may be a therapeutic option after discontinuation of teriparatide therapy.
Asunto(s)
Conservadores de la Densidad Ósea/farmacología , Densidad Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Ligando RANK/antagonistas & inhibidores , Teriparatido/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Resorción Ósea , Femenino , Ratones , Ratones Endogámicos C57BL , Osteogénesis/efectos de los fármacos , OvariectomíaRESUMEN
Osteoclasts resorb bone by attaching on the bone matrix and forming a sealing zone. In Src-deficient mice, osteoclasts cannot form the actin ring, a characteristic actin structure that seals the resorbed area, and resorb hardly any bone as a result. However, the molecular mechanism underlying the role of Src in the regulation and organization of the actin ring is still unclear. We identified an actin-regulatory protein, protein phosphatase 1 regulatory subunit 18 (PPP1r18), as an Src-binding protein in an Src-, Yes-, and Fyn-deficient fibroblast (SYF) cell line overexpressing a constitutively active form of Src. PPP1r18 was localized in the nucleus and actin ring. PPP1r18 overexpression in osteoclasts inhibited terminal differentiation, actin ring formation, and bone-resorbing activity. A mutation of the protein phosphatase 1 (PP1)-binding domain of PPP1r18 rescued these phenotypes. In contrast, PPP1r18 knockdown promoted terminal differentiation and actin ring formation. In summary, we showed that PPP1r18 likely plays a role in podosome organization and bone resorption.
Asunto(s)
Actinas/metabolismo , Resorción Ósea/metabolismo , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Osteoclastos/citología , Osteoclastos/metabolismo , Animales , Adhesión Celular/fisiología , Diferenciación Celular/fisiología , Línea Celular , Humanos , Masculino , Ratones , Ratones Transgénicos , Unión Proteica , Proteína Fosfatasa 1/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Neuropéptido Y/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
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.