Osteoclastic resorptive capacity is suppressed in patients receiving hyperbaric oxygen therapy

Background and purpose — Hypoxia, necrosis, and bone loss are hallmarks of many skeletal diseases. Hyperbaric oxygen therapy (HBO) is often used as an adjunctive therapy in these cases. However the in vivo effect of HBO on osteoclast formation has not been fully established. We therefore carried out a longitudinal study to examine the effect of HBO on osteoclast formation and bone resorptive capacity in patients who were referred to the Plymouth Hyperbaric Medical Centre.

Methods — Osteoclast precursors were isolated from peripheral blood prior to and following 10 and 25 daily hyperbaric treatments (100% O₂ at 2.4 atmospheres absolute ATA for 90 min) to determine osteoclast formation and resorptive capacity. The expression of key regulators of osteoclast differentiation RANK, Dc-STAMP, and NFATc1 was also assessed by quantitative real-time PCR.

Results — HBO reduced the ability of precursors to form osteoclasts and reduced bone resorption in a treatment-dependent manner. The initial suppressive effect of HBO was more pronounced on mononuclear osteoclast formation than on multinuclear osteoclast formation, and this was accompanied by reduction in the expression of key regulators of osteoclast formation, RANK and Dc-STAMP.

Interpretation — This study shows for the first time that in vivo, HBO suppresses the ability of monocytic precursors to form resorptive osteoclasts.     

Intracranial pressure responses during hyperbaric oxygen therapy.

The responses of intracranial pressure (ICP) to hyperbaric oxygen (HBO) therapy and arterial gas pressures were investigated. ICP was measured through a ventricular or spinal drainage catheter in patients with brain tumor or cerebrovascular disease. Changes in ICP, heart rate (HR), arterial blood pressure (ABP), and transcutaneous partial pressure of carbon dioxide (PtcCO2) or oxygen (PtcO2) were recorded continuously during air or 100% O2 breathing at 1 and 2.5 atmospheres absolute (ATA). HR and PtcCO2 decreased and mean ABP was unchanged during HBO inhalation. ICP was reduced at the beginning and tended to increase gradually during HBO inhalation. The change from air to O2 without altering respiratory frequency and volume caused a gradual increase of ICP and PtcCO2 with a transient ICP reduction in an artificially respirated patient. Intentionally reduced respiration to maintain PtcCO2 at the value at 2.5 ATA with air caused the ICP to return to near the value at 2.5 ATA with air even during HBO inhalation. These findings suggest that reduced ICP is initially due to direct cerebral vasoconstriction caused by hyperoxia and is maintained mainly by induced hypocapnia during HBO inhalation. Care is required when giving HBO therapy to patients with a high ICP and/or who are respirated artificially.     

Dihydroartemisinin,an Anti‐Malaria Drug,Suppresses Estrogen Deficiency‐Induced Osteoporosis,Osteoclast Formation,and RANKL‐Induced Signaling Pathways

Osteoporosis is an osteolytic disease that features enhanced osteoclast formation and bone resorption. Identification of agents that can inhibit osteoclast formation and function is important for the treatment of osteoporosis. Dihydroartemisinin is a natural compound used to treat malaria but its role in osteoporosis is not known. Here, we found that dihydroartemisinin can suppress RANKL‐induced osteoclastogenesis and bone resorption in a dose‐dependent manner. Dihydroartemisinin inhibited the expression of osteoclast marker genes such as cathepsin K, calcitonin receptor, and tartrate‐resistant acid phosphatase (TRAcP). Furthermore, dihydroartemisinin inhibited RANKL‐induced NF‐κB and NFAT activity. In addition, using an in vivo ovariectomized mouse model, we show that dihydroartemisinin is able to reverse the bone loss caused by ovariectomy. Together, this study shows that dihydroartemisinin attenuates bone loss in ovariectomized mice through inhibiting RANKL‐induced osteoclast formation and function. This indicates that dihydroartemisinin, the first physiology or medicine nobel prize discovery of China, is a potential treatment option against osteolytic bone disease. © 2015 American Society for Bone and Mineral Research.     

A Bone Anabolic Effect of RANKL in a Murine Model of Osteoporosis Mediated Through FoxP3+ CD8 T Cells

TNF‐α and IL‐17 secreted by proinflammatory T cells (T_EFF) promote bone erosion by activating osteoclasts. We previously demonstrated that in addition to bone resorption, osteoclasts act as antigen‐presenting cells to induce FoxP3 in CD8 T cells (Tc_REG). The osteoclast‐induced regulatory CD8 T cells limit bone resorption in ovariectomized mice (a murine model of postmenopausal osteoporosis). Here we show that although low‐dose receptor activator of NF‐κB ligand (RANKL) maximally induces Tc_REG via Notch signaling pathway to limit bone resorption, high‐dose RANKL promotes bone resorption. In vitro, both TNF‐α and IL‐17, cytokines that are abundant in ovariectomized animals, suppress Tc_REG induction by osteoclasts by repressing Notch ligand expression in osteoclasts, but this effect can be counteracted by addition of RANKL. Ovariectomized mice treated with low‐dose RANKL induced Tc_REG that suppressed bone resorption, decreased T_EFF levels, and increased bone formation. High‐dose RANKL had the expected osteolytic effect. Low‐dose RANKL administration in ovariectomized mice lacking CD8 T cells was also osteolytic, confirming that Tc_REG mediate this bone anabolic effect. Our results show that although RANKL directly stimulates osteoclasts to resorb bone, it also controls the osteoclasts' ability to induce regulatory T cells, engaging an important negative feedback loop. In addition to the conceivable clinical relevance to treatment of osteoporosis, these observations have potential relevance to induction of tolerance and autoimmune diseases. © 2015 American Society for Bone and Mineral Research.     

Preconditioning with hyperbaric oxygen and hyperoxia induces tolerance against spinal cord ischemia in rabbits

BACKGROUND: The aim of this study was to determine if the ischemic tolerance could be induced in the spinal cord by pretreatment with hyperbaric oxygen (HBO) and what components of HBO (hyperoxia, hyperbaricity, and combination of these two) were critical in the induction of tolerance against ischemic injury. METHODS: In experiment 1, 21 rabbits were randomly assigned to one of three groups (n = 7 each): animals in the control group received no HBO before spinal cord ischemia; animals in the HBO-1 and HBO-2 groups received HBO (2.5 atmosphere absolute [ATA], 100% O2) pretreatment 1 h/day for 3 and 5 days before ischemia, respectively. In experiment 2, 48 rabbits were randomly assigned to one of four groups (n = 12 each): the control group received no HBO (21% O2, 1 ATA, 1 h/day, 5 days) before spinal cord ischemia; the HB group received 1-h treatment in 21% O2 at 2.5 ATA each day for 5 days; the HO group received 1-h treatment in 100% oxygen at 1 ATA each day for 5 days; and the HBO group received HBO (2.5 ATA, 100% O2) treatment 1 h/day for 5 days. Twenty-four hours after the last treatment, spinal cord ischemia was induced by an infrarenal aorta clamping for 20 min. Forty-eight hours after reperfusion, hind-limb motor function and histopathology of the spinal cord were examined in a blinded fashion. RESULTS: In experiment 1, the neurologic outcome in the HBO-2 group was better than that of the control group (P = 0.004). The number of normal neurons in the anterior spinal cord in the HBO-2 group was more than that of the control group (P = 0.021). In experiment 2, the neurologic and histopathologic outcomes in the HBO group were better than that of the control group (P < 0.01). The histopathologic outcome in the HO group was better than that in the control group (P < 0.05). CONCLUSIONS: Serial exposure to high oxygen tension induced ischemic tolerance in spinal cord of rabbits. Simple hyperbaricity (2.5 ATA, 21% O2) did not induce ischemic tolerance.     

Preconditioning with Hyperbaric Oxygen and Hyperoxia Induces Tolerance against Spinal Cord Ischemia in Rabbits

Background: The aim of this study was to determine if the ischemic tolerance could be induced in the spinal cord by pretreatment with hyperbaric oxygen (HBO) and what components of HBO (hyperoxia, hyperbaricity, and combination of these two) were critical in the induction of tolerance against ischemic injury.

Methods: In experiment 1, 21 rabbits were randomly assigned to one of three groups (n = 7 each): animals in the control group received no HBO before spinal cord ischemia; animals in the HBO-1 and HBO-2 groups received HBO (2.5 atmosphere absolute [ATA], 100% O2) pretreatment 1 h/day for 3 and 5 days before ischemia, respectively. In experiment 2, 48 rabbits were randomly assigned to one of four groups (n = 12 each): the control group received no HBO (21% O2, 1 ATA, 1 h/day, 5 days) before spinal cord ischemia; the HB group received 1-h treatment in 21% O2 at 2.5 ATA each day for 5 days; the HO group received 1-h treatment in 100% oxygen at 1 ATA each day for 5 days; and the HBO group received HBO (2.5 ATA, 100% O2) treatment 1 h/day for 5 days. Twenty-four hours after the last treatment, spinal cord ischemia was induced by an infrarenal aorta clamping for 20 min. Forty-eight hours after reperfusion, hind-limb motor function and histopathology of the spinal cord were examined in a blinded fashion.

Results: In experiment 1, the neurologic outcome in the HBO-2 group was better than that of the control group (P = 0.004). The number of normal neurons in the anterior spinal cord in the HBO-2 group was more than that of the control group (P = 0.021). In experiment 2, the neurologic and histopathologic outcomes in the HBO group were better than that of the control group (P < 0.01). The histopathologic outcome in the HO group was better than that in the control group (P < 0.05).     

Effects of different exposures of hyperbaric oxygen on ligament healing in rats.

Hyperbaric oxygen (HBO) is a method of augmenting, intermittently, oxygen availability to tissues. We examined the effect of three different HBO exposures on the healing of experimentally induced ligament lacerations in the right hind limb of 44 male Wistar rats. Animals were divided into four groups after ligament injury: (a) control group, animals breathed room air at 1 ATA (atmosphere absolute) in a hyperbaric chamber for 60 min; (b) HBO treatment at 1.5 ATA for 30 min once a day, (c) HBO treatment at 2 ATA for 30 min once a day, (d) 2 ATA for 60 min once a day. At 14 days post-ligament injury, we compared the ligaments of the four treatment groups for gross appearance, histology and expression of pro-alpha(I) mRNA by northern hybridization. Our results indicate that HBO was effective in promoting ligament healing compared to control (p < 0.01). Of these three exposures, HBO at 2 ATA for 60 min was the most effective, resulting in enhanced extra-cellular matrix deposition as measured by collagen synthesis.     

Urinary excretion rate of methylguanidine as a new maker of active oxygen in vivo: demonstration in hyperbaric oxygen therapy

Methylguanidine (MG) which is known as a uremic toxin, is synthesized from creatinine (Cre). We have clarified that active oxygen plays an important role on MG synthesis in vitro and in rat hepatocytes. On the other hand, hyperoxia is very injurious in various tissues, and it has been reported that active oxygen produced in hyperoxia plays an important role on the tissue injury. This study was performed to investigate the effect of hyperoxia on MG synthesis in vivo. The subjects in this study were patients who were treated by hyperbaric oxygen therapy (HBO). Serum Cre, MG, and urinary Cre, MG before and after HBO were measured in these subjects. The subjects were classified into four groups. Group I-III were undergone HBO with condition of 100% O2, 2 atmosphere absolute (ATA), 1 hour, (I: Ccr less than 10 ml/min, II: 10 less than or equal to Ccr less than 50 ml/min, III: Ccr greater than or equal to 50 ml/min) and group IV (Ccr greater than or equal to 50 ml/min) with 100%O2, 3ATA, 1 hour. Urinary excretion rate of MG (urine MG/urine Cre) significantly increased after HBO therapy in every group. Urine MG/urine Cre/serum Cre ratio which was used as a index of MG synthesis rate also increased. In this study, it is clarified that MG excretion rate increases in hyperoxic condition. These results suggest that active oxygen plays an important role on MG synthesis in vivo, and that the urine MG/urine Cre/serum Cre ratio can be a useful maker of the active oxygen products in vivo.     

Ferric ion could facilitate osteoclast differentiation and bone resorption through the production of reactive oxygen species

Iron overload is widely regarded as a risk factor for osteoporosis. It has been demonstrated that iron can inhibit osteoblast differentiation. However, the effects of iron on osteoclast differentiation and bone resorption remain controversial. In this study, we found that ferric ion promoted Receptor Activator of Nuclear Factor κ B Ligand (RANKL)‐induced osteoclast (OC) formation in both RAW264.7 cells and bone marrow‐derived macrophages (BMMs), and this effect was accompanied by elevated levels of reactive oxygen species (ROS) and oxidative stress. Moreover, this effect was attenuated by the administration of antioxidant N‐acetyl‐L ‐cysteine (NAC). Therefore, we conclude that ferric ion can promote osteoclast differentiation and bone resorption through the production of ROS. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1843–1852, 2012     

Hyperbaric oxygen therapy in a mouse model of implant‐associated osteomyelitis

Implant associated osteomyelitis (OM) is difficult to treat with antibiotics, and outcomes remain poor. Some reports suggest that hyperbaric oxygen treatment is a safe and effective means of treating OM. We tested this hypothesis in a murine model. Clinical isolates of methicillin‐resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Klebsiella pneumoniae were used. The mice were infected with each of the three pathogens, treated with 100% oxygen at high pressure, hyperbaric oxygen (HBO), and monitored for the ability of HBO to prevent and/or clear the OM infection. Assessments included bacterial burden of the tibias and lesion scores, as well as receptor activator of NF‐κB ligand (RANKL) and myeloperoxidase (MPO) concentrations. HBO resulted in more severe lesion scores and higher RANKL and MPO concentrations for MRSA. A significant positive correlation was found between RANKL concentration and lesion score. No significant difference was found with HBO in P. aeruginosa infections and K. pneumoniae seems to either not infect bone well or get cleared before establishing an infection. The model is useful for studying OM infections caused by MRSA and P. aeruginosa, but HBO does not appear to be an efficacious treatment of an implant‐associated OM infection. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:203–208, 2012     

Denosumab,a Fully Human Monoclonal Antibody to RANKL,Inhibits Bone Resorption and Increases BMD in Knock‐In Mice That Express Chimeric (Murine/Human) RANKL

RANKL is a TNF family member that mediates osteoclast formation, activation, and survival by activating RANK. The proresorptive effects of RANKL are prevented by binding to its soluble inhibitor osteoprotegerin (OPG). Recombinant human OPG‐Fc recognizes RANKL from multiple species and reduced bone resorption and increased bone volume, density, and strength in a number of rodent models of bone disease. The clinical development of OPG‐Fc was discontinued in favor of denosumab, a fully human monoclonal antibody that specifically inhibits primate RANKL. Direct binding assays showed that denosumab bound to human RANKL but not to murine RANKL, human TRAIL, or other human TNF family members. Denosumab did not suppress bone resorption in normal mice or rats but did prevent the resorptive response in mice challenged with a human RANKL fragment encoded primarily by the fifth exon of the RANKL gene. To create mice that were responsive to denosumab, knock‐in technology was used to replace exon 5 from murine RANKL with its human ortholog. The resulting “huRANKL” mice exclusively express chimeric (human/murine) RANKL that was measurable with a human RANKL assay and that maintained bone resorption at slightly reduced levels versus wildtype controls. In young huRANKL mice, denosumab and OPG‐Fc each reduced trabecular osteoclast surfaces by 95% and increased bone density and volume. In adult huRANKL mice, denosumab reduced bone resorption, increased cortical and cancellous bone mass, and improved trabecular microarchitecture. These huRANKL mice have potential utility for characterizing the activity of denosumab in a variety of murine bone disease models.     

Hyperbaric hyperoxia suppresses growth of Staphylococcus aureus, including methicillin-resistant strains

Purpose. We investigated the effects of increased oxygen tension on the in vitro growth of Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), and Escherichia coli (E. coli). Methods. The effects of oxygen tension [normobaric normoxia (21% O₂ at 1 atm), normobaric hyperoxia (100% O₂ at 1 atm), hyperbaric normoxia (21% O₂ at 2 atm), and hyperbaric hyperoxia (100% O₂ at 2 atm) on the in vitro growth of MRSA, MSSA, and E. coli were investigated by population analysis. Results. Compared with normobaric normoxia, a 90-min exposure to hyperbaric hyperoxia significantly inhibited growth of both MRSA (by 25.0 ± 3.0%, mean ± SEM; P < 0.01) and MSSA (by 24.0 ± 3.3%; P < 0.01). Normobaric hyperoxia and hyperbaric normoxia were without effect. In contrast, the growth of E. coli was not affected by any of the above treatments. Conclusion. Our results indicate that the bacterium S. aureus, including resistant strains, is susceptible to oxygen stress. The observation that relatively brief (90-min) treatment with hyperbaric hyperoxia is sufficient to produce significant growth inhibition suggests that hyperbaric hyperoxia may be useful in the treatment of serious staphylococcal infections. Received: March 6, 2000 / Accepted: August 7, 2000     

Selective tyrosine kinase inhibition of insulin‐like growth factor‐1 receptor inhibits human and mouse breast cancer–induced bone cell activity,bone remodeling,and osteolysis

Insulin‐like growth factor 1 (IGF‐1) plays an important role in both bone metabolism and breast cancer. In this study, we investigated the effects of the novel IGF‐1 receptor tyrosine kinase inhibitor cis‐3‐[3‐(4‐methyl‐piperazin‐l‐yl)‐cyclobutyl]‐1‐(2‐phenyl‐quinolin‐7‐yl)‐imidazo[1,5‐a]pyrazin‐8‐ylamine (PQIP) on osteolytic bone disease associated with breast cancer. Human MDA‐MB‐231 and mouse 4T1 breast cancer cells enhanced osteoclast formation in receptor activator of NF‐κB ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF) stimulated bone marrow cultures, and these effects were significantly inhibited by PQIP. Functional studies in osteoclasts showed that PQIP inhibited both IGF‐1 and conditioned medium–induced osteoclast formation by preventing phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (Akt) activation without interfering with RANKL or M‐CSF signaling. Treatment of osteoblasts with PQIP significantly inhibited the increase in RANKL/osteoprotegerin (OPG) ratio by IGF‐1 and conditioned medium and totally prevented conditioned medium–induced osteoclast formation in osteoblast–bone marrow (BM) cell cocultures, thereby suggesting an inhibitory effect on osteoblast–osteoclast coupling. PQIP also inhibited IGF‐1–induced osteoblast differentiation, spreading, migration, and bone nodule formation. Treatment with PQIP significantly reduced MDA‐MB‐231 conditioned medium–induced osteolytic bone loss in a mouse calvarial organ culture system ex vivo and in adult mice in vivo. Moreover, once daily oral administration of PQIP significantly decreased trabecular bone loss and reduced the size of osteolytic bone lesions following 4T1 intratibial injection in mice. Quantitative histomorphometry showed a significant reduction in bone resorption and formation indices, indicative of a reduced rate of cancer‐associated bone turnover. We conclude that inhibition of IGF‐1 receptor tyrosine kinase activity by PQIP suppresses breast cancer–induced bone turnover and osteolysis. Therefore, PQIP, and its novel derivatives that are currently in advanced clinical development for the treatment of a number of solid tumors, may be of value in the treatment of osteolytic bone disease associated with breast cancer. © 2013 American Society for Bone and Mineral Research.     

Hyperbaric oxygen treatment suppresses MAPK signaling and mitochondrial apoptotic pathway in degenerated human intervertebral disc cells

Nucleus pulposus cells (NPCs) from degenerating discs produce catabolic and inflammatory factors, including interleukin (IL)‐1 and nitric oxide (NO). Enhanced production of NO has been implicated in the apoptosis of degenerating disc cells. This study evaluates the effects of hyperbaric oxygen (HBO) on degenerated human NPCs. All hyperoxic cells were exposed to 100% O₂ at 2.5 atmospheres absolute (ATA). Phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK1/2), c‐Jun N‐terminal kinase (JNK), and p38 mitogen‐activated protein kinase (MAPK) in NPCs was detected using the phosphor‐kinase array kit. RNA was isolated for real‐time polymerase chain reaction (PCR) analysis of aggrecan and type II collagen gene expression. The levels of IL‐1β and NO were quantified by enzyme‐linked immunosorbent assay (ELISA). To identify the HBO‐induced anti‐apoptotic pathways, expression of Bcl‐2 and Bax proteins as well as activation of cysteine‐containing aspartate‐specific proteases (caspases) 3, 8, and 9 was evaluated using Western blotting after HBO treatment. Our data showed that HBO treatment decreased the expression of IL‐1β, suppressed phosphorylation of ERK1/2, JNK, and p38 MAPK, decreased synthesis of NO, and increased the gene expression of aggrecan and type II collagen in NPCs as compared with the atmospheric treatment. HBO up‐regulated the ratio of Bcl‐2 to Bax expression and reduced the activity of caspases 9 and 3 but not of caspase 8, indicating a selective effect over the mitochondrial apoptosis pathway in degenerated NPCs. These results support our hypothesis that HBO treatment suppresses MAPK signaling and mitochondrial apoptotic pathway in degenerated human intervertebral disc cells. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 204–209, 2013     

Regulation of RANKL‐induced osteoclastogenesis by RING finger protein RNF114

Normal bone remodeling is a continuous process orchestrated by bone‐resorbing osteoclasts and bone‐forming osteoblasts, which an imbalance in bone remodeling results in metabolic bone diseases. RANKL, a member of the TNF cytokine family, functions as a key stimulator for osteoclast differentiation and maturation. Here, we report that RNF114, previously identified as a psoriasis susceptibility gene, plays a regulatory role in the RANKL/RANK/TRAF6 signaling pathway that mediates osteoclastogenesis. Our results demonstrated that RNF114 expression was significantly down‐regulated in mouse osteoclast precursor cells undergoing RANKL‐induced osteoclast differentiation. RNF114 knockout did not affect development or viability of the subpopulation of bone marrow macrophages capable of differentiating into osteoclasts in culture. However, in the presence of RANKL, RNF114 knockout bone marrow macrophages exhibited enhanced cell proliferation and augmented osteoclast differentiation, as shown by an increased expression of mature osteoclast markers, increased osteoclastic TRAP activity and bone resorption. Conversely, ectopic expression of RNF114 inhibited CTSK expression, TRAP activity, and bone resorption in RANKL‐treated pre‐osteoclasts. RNF114 also suppressed RANKL‐activated NFATc1 expression and NFAT‐regulated promoter activity. RNF114 suppressed TRAF6‐, but not TAK1/TAB2‐mediated NF‐κB activation downstream of RANKL/RANK. In particular, TRAF6 protein levels were down‐regulated by RNF114, possibly via K48‐mediated proteasome‐dependent degradation. These data suggested that RNF114's inhibitory effect on RANKL‐stimulated osteoclastogenesis was mediated by blocking RANK/TRAF6/NF‐κB signal transduction. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:159–166, 2018.     

Effect of different periods of hyperbaric oxygen on ischemia-reperfusion injury of rat skeletal muscle

The effect of hyperbaric oxygen (HBO) on ischemia-reperfusion injury of skeletal muscle, applied during different periods, was studied in 56 male rats. Animals were subjected to 6-h ischemia by a tourniquet over the major femoral trocanter and 4 (A) or 24 (B) h of reperfusion. HBO was carried out during 1 h in an acrylic chamber at a pressure of 2.0 ATA (100% oxygen): in the last 60 min of ischemia (II), after ischemia, during 1-h reperfusion time (III), and during the last hour of ischemia plus 1-h reperfusion (IV). Group I was the control group. After 4- or 24-h reperfusion, samples of the soleus muscle were stained by H&E and analyzed immunohistochemically. No interstitial hemorrhage, neutrophil infiltrate, or cellular necrosis were induced by HBO. The apoptosis index did not differ among the groups. HBO reduced morphologic alterations and promoted better results when administered in the ischemia plus reperfusion period (GIV).     

Menatetrenone (Vitamin K<Subscript>2</Subscript>) Acts Directly on Circulating Human Osteoclast Precursors

It is still not certain what the direct effect of menatetrenone is on osteoclast precursors. In the present study, we investigated whether menatetrenone has a direct effect on circulating osteoclast precursors to influence osteoclast differentiation. Monocytes isolated from human peripheral blood were cultured with receptor-activated NF-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Menatetrenone or vitamin K₁ was then added to the cultures. Geranylgeraniol or phytol (the respective side chain) was also added to the cultures instead of menatetrenone or vitamin K₁, respectively. After 7 and 14 days incubation, cultures were evaluated for cytochemical and functional evidence of osteoclast formation. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) and the percentage area of lacunar resorption induced by RANKL and M-CSF were decreased when menatetrenone or geranylgeraniol was added to the cultures. Dose-dependent inhibition of osteoclast formation and lacunar resorption was seen when the cultures were treated with menatetrenone or geranylgeraniol. In contrast, vitamin K₁ or phytol did not affect the number of TRAP-positive MNCs nor the percentage area of lacunar resorption. These results indicate that menatetrenone not only influences osteoclast formation via bone stromal cells but also acts directly on circulating osteoclast precursors to influence osteoclast differentiation. These findings also suggest that geranylgeraniol, the side chain of menatetrenone, plays an important role in this inhibitory effect.     

Siglec‐15 Regulates Osteoclast Differentiation by Modulating RANKL‐Induced Phosphatidylinositol 3‐Kinase/Akt and Erk Pathways in Association With Signaling Adaptor DAP12

Siglecs are a family of sialic acid–binding immunoglobulin‐like lectins that regulate the functions of cells in the innate and adaptive immune systems through glycan recognition. Here we show that Siglec‐15 regulates osteoclast development and bone resorption by modulating receptor activator of nuclear factor κB ligand (RANKL) signaling in association with DNAX‐activating protein 12 kDa (DAP12), an adaptor protein bearing an immunoreceptor tyrosine‐based activation motif (ITAM). Among the known Siglecs expressed in myeloid lineage cells, only Siglec‐15 was upregulated by RANKL in mouse primary bone marrow macrophages. Siglec‐15–deficient mice exhibit mild osteopetrosis resulting from impaired osteoclast development. Consistently, cells lacking Siglec‐15 exhibit defective osteoclast development and resorptive activity in vitro. RANKL‐induced activation of phosphatidylinositol 3‐kinase (PI3K)/Akt and Erk pathways were impaired in Siglec‐15–deficient cells. Retroviral transduction of Siglec‐15–null osteoclast precursors with wild‐type Siglec‐15 or mutant Siglec‐15 revealed that the association of Siglec‐15 with DAP12 is involved in the downstream signal transduction of RANK. Furthermore, we found that the ability of osteoclast formation is preserved in the region adjacent to the growth plate in Siglec‐15–deficient mice, indicating that there is a compensatory mechanism for Siglec‐15–mediated osteoclastogenesis in the primary spongiosa. To clarify the mechanism of this compensation, we examined whether osteoclast‐associated receptor (OSCAR)/Fc receptor common γ (FcRγ) signaling, an alternative ITAM‐mediated signaling pathway to DAP12, rescues impaired osteoclastogenesis in Siglec‐15–deficient cells. The ligands in type II collagen activate OSCAR and rescue impaired osteoclastogenesis in Siglec‐15–deficient cells when cultured on bone slices, indicating that Siglec‐15–mediated signaling can be compensated for by signaling activated by type II collagen and other bone matrix components in the primary spongiosa. Our findings indicate that Siglec‐15 plays an important role in physiologic bone remodeling by modulating RANKL signaling, especially in the secondary spongiosa. © 2013 American Society for Bone and Mineral Research.     

Inhibition of osteoclastogenesis by RNA interference targeting RANK

ABSTRACT: BACKGROUND: Osteoclasts and osteoblasts regulate bone resorption and formation to allow bone remodeling and homeostasis. The balance between bone resorption and formation is disturbed by abnormal recruitment of osteoclasts. Osteoclast differentiation is dependent on the receptor activator of nuclear factor NF-kappa B (RANK) ligand (RANKL) as well as the macrophage colony-stimulating factor (M-CSF). The RANKL/RANK system and RANK signaling induce osteoclast formation mediated by various cytokines. The RANK/RANKL pathway has been primarily implicated in metabolic, degenerative and neoplastic bone disorders or osteolysis. The central role of RANK/RANKL interaction in osteoclastogenesis makes RANK an attractive target for potential therapies in treatment of osteolysis. The purpose of this study was to assess the effect of inhibition of RANK expression in mouse bone marrow macrophages on osteoclast differentiation and bone resorption. METHODS: Three pairs of short hairpin RNAs (shRNA) targeting RANK were designed and synthesized. The optimal shRNA was selected among three pairs of shRNAs by RANK expression analyzed by Western blot and Real-time PCR. We investigated suppression of osteoclastogenesis of mouse bone marrow macrophages (BMMs) using the optimal shRNA by targeting RANK. RESULTS: Among the three shRANKs examined, shRANK-3 significantly suppressed [88.3%] the RANK expression (p < 0.01). shRANK-3 also brought about a marked inhibition of osteoclast formation and bone resorption as demonstrated by tartrate--resistant acid phosphatase (TRAP) staining and osteoclast resorption assay. The results of our study show that retrovirus-mediated shRANK-3 suppresses osteoclast differentiation and osteolysis of BMMs. CONCLUSIONS: These findings suggest that retrovirus-mediated shRNA targeting RANK inhibits osteoclast differentiation and osteolysis. It may appear an attractive target for preventing osteolysis in humans with a potential clinical application.