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Summary Objectives Orthodontic tooth movement (OTM) increases sympathetic and sensory neurological markers in periodontal tissue. However, the relationship between the sympathetic and sensory nervous systems during OTM remains unclear. Therefore, the present study investigated the relationship between the sympathetic and sensory nervous systems activated by OTM using pharmacological methods. Materials and Methods We compared the effects of sympathectomy and sensory nerve injury during OTM in C57BL6/J mice. Capsaicin (CAP) was used to induce sensory nerve injury. Sympathectomy was performed using 6-hydroxydopamine. To investigate the effects of a β-agonist on sensory nerve injury, isoproterenol (ISO) was administered to CAP-treated mice. Furthermore, to examine the role of the central nervous system in OTM, the ventromedial hypothalamic nucleus (VMH) was ablated using gold thioglucose. Results Sensory nerve injury and sympathectomy both suppressed OTM and decreased the percent of the alveolar socket covered with osteoclasts (Oc.S/AS) in periodontal tissue. Sensory nerve injury inhibited increases in OTM-induced calcitonin gene-related peptide (CGRP) immunoreactivity (IR), a marker of sensory neurons, and tyrosine hydroxylase (TH) IR, a marker of sympathetic neurons, in periodontal tissue. Although sympathectomy did not decrease the number of CGRP-IR neurons in periodontal tissue, OTM-induced increases in the number of TH-IR neurons were suppressed. The ISO treatment restored sensory nerve injury-inhibited tooth movement and Oc.S/AS. Furthermore, the ablation of VMH, the centre of the sympathetic nervous system, suppressed OTM-induced increases in tooth movement and Oc.S/AS. Conclusions The present results suggest that OTM-activated sensory neurons contribute to enhancements in osteoclast activity and tooth movement through sympathetic nervous signalling.

Background/Aim: Glycosphingolipids are known to be involved in bone metabolism. However, their roles and regulatory mechanisms in osteoblast proliferation are largely unknown. In this study, we examined the effects of inhibitors of glucosylceramide synthase (GCS), which is responsible for the generation of all glycosphingolipids, on osteoblast proliferation. Materials and Methods: We analyzed the expression of glycosphingolipids and cell growth in MC3T3-E1 mouse osteoblast cells treated with the GCS inhibitors miglustat, D-PDMP and D-PPMP. We also conducted microarray analysis and RNA interference to identify genes involved in cell growth regulated by GCS. Results: Glycosphingolipids GD1a and Gb4 expressed in MC3T3-E1 cells, were suppressed by GCS inhibitors. Furthermore, the proliferation of MC3T3-E1 cells was suppressed by the inhibitors. Using microarray analysis, we predicted nine genes (Fndc1, Acta2, Igfbp5, Cox6a2, Cth, Mymk, Angptl6, Mab21l2, and Igsf10) suppressed by all three inhibitors. Furthermore, partial silencing of Angptl6 by RNA interference reduced MC3T3-E1 cell growth. Conclusion: These results show that GCS regulates proliferation through Angptl6 in osteoblasts.

Objective Regulation of bone metabolism by the sympathetic nervous system has recently been clarified. Tooth movement is increased by increased bone metabolic turnover due to sympathetic activation. This study aimed to compare the effects of the β-adrenergic receptor (β-AR) blockers atenolol (β1-AR blocker), butoxamine (β2-AR blocker) and propranolol (non-selective β-AR blocker) on tooth movement in spontaneously hypertensive rats (SHR) with sympathicotonia. Materials and methods Spontaneously hypertensive rats were divided into the following four groups: an SHR control group and groups treated with 0.1 mg/kg atenolol, 1 mg/kg butoxamine or 1 mg/kg propranolol (n = 6 rats/group). Atenolol, butoxamine or propranolol was administered daily to each treatment group, and orthodontic force was applied using a closed-coil spring. Finally, immunohistochemical analysis was performed for receptor activator of nuclear factor kappa-B ligand (RANKL) and sclerostin (SOST). Results Atenolol, butoxamine and propranolol inhibited tooth movement and increased maxillary alveolar bone volume. Histological analysis revealed that these β-AR blockers decreased osteoclast activity on the compression side. Furthermore, immunohistochemical analysis revealed that atenolol, butoxamine and propranolol decreased the number of RANKL- and SOST-positive osteocytes on the compression side. Conclusions β-AR blockers decreased tooth movement and downregulated SOST in osteocytes, accompanied by increasing alveolar bone resorption.

Aims Opioid receptor blockers such as naloxone and naltrexone have been suggested to have a bone mass-increasing effect. However, the mechanisms at play have not been clarified. We examined the effects of naltrexone on osteoblasts and determined the expression of opioid growth factor receptor (OGFR) in osteoblasts. Naltrexone blocks the OGFR and other canonical opioid receptors. Thus, we designed experiments to clarify the effects of naltrexone on bone tissue by examining the physiological role of OGFR signaling in osteoblasts and the changes in bone structure after naltrexone systemic administration in mice. Main methods We used mouse osteoblast-like cell line MC3T3-E1 for in vitro experiments. We cultured MC3T3-E1 cells in the presence of the OGFR agonist met-enkephalin (met-enk). Then, we measured cell proliferation activity and analyzed the expression levels of cell proliferation-related genes. For our in vivo experiments, we administered naltrexone intraperitoneally to mice daily for 28 days and administered the animals in the control group equivalent volumes of saline. After sacrificing the mice, we performed micro-computed tomography and bone morphology analyses. Key findings Met-enk suppressed cell proliferation in MC3T3-E1 cells. Moreover, Low dose naltrexone administration significantly increased their femoral bone mass, bone formation ratio, and osteoblast number/bone surface values when comparing the values for the same variables in the control group. Significance Our results suggest that naltrexone increases bone mass due to osteoblast number increments caused by the OGFR signaling block. Opioid receptor blockers have potential as therapeutic agents for osteoporosis as well as opioid antagonists.

Introduction Human periodontal ligament mesenchymal stem cells (hPDLMSCs) have been known that they play important roles in homeostasis and regeneration of periodontal tissues. Additionally, spheroids are superior to monolayer-cultured cells. We investigated the characteristics and potential of periodontal tissue regeneration in co-cultured spheroids of hPDLMSCs and human umbilical vein endothelial cells (HUVECs) in vitro and in vivo. Methods Co-cultured spheroids were prepared with cell ratios of hPDLMSCs: HUVECs = 1:1, 1:2, and 2:1, using microwell chips. Real-time polymerase chain reaction (PCR) analysis, Enzyme-Linked Immuno Sorbent Assay (ELISA), and nodule formation assay were performed to examine the properties of co-cultured spheroids. Periodontal tissue defects were prepared in the maxillary first molars of rats and subjected to transplantation assay. Results The expression levels of stemness markers, vascular endothelial growth factor (VEGF), osteogenesis-related genes were up-regulated in co-cultured spheroids, compared with monolayer and spheroid-cultured hPDLMSCs. The nodule formation was also increased in co-cultured spheroids, compared with monolayer and spheroid cultures of hPDLMSCs. Treatment with co-cultured spheroids enhanced new cementum formation after 4 or 8 weeks of transplantation, although there was no significant difference in the new bone formation between co-cultured spheroids and hPDLMSC spheroids. Conclusions We found that co-cultured spheroids enhance the periodontal tissue regeneration. Co-cultured spheroids of hPDLMSCs and HUVECs may be a useful therapy that can induce periodontal tissue regeneration.

Dental pulp is known to play crucial roles in homeostasis of teeth and periodontal tissue. Although resorption of bone around the roots of nonvital teeth is occasionally observed in clinical practice, little is known about the role of dental pulp in osteoclastogenesis. Here we evaluated the effects of conditioned medium (CM) from rat dental pulp on osteoclastogenesis. It was found that the CM reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts, but did not alter the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 and TRAP. To further understand the mechanism behind these results, we evaluated the effects of CM on osteoclast precursors and found that the CM removed cell processes, resulting in a significant reduction in the number of attached cells and an increase in the number of freely floating cells. Furthermore, the CM suppressed the mRNA levels of focal adhesion kinase and paxillin, which are involved in cell adhesiveness and spreading. Collectively, the present results show that CM from dental pulp serves as an inhibitor of osteoclastogenesis by reducing the number and adhesiveness of osteoclast precursors, suggesting novel therapeutic applicability for osteoporosis.

Recent studies have revealed that the sensory nervous system is involved in bone metabolism. However, the mechanism of communication between neurons and osteoblasts is yet to be elucidated. In this study, we investigated the signaling pathways between sensory neurons of the dorsal root ganglion (DRG) and the osteoblast-like MC3T3-E1 cells using an in vitro coculture system. Our findings indicate that signal transduction from DRG-derived neurons to MC3T3-E1 cells is suppressed by antagonists of the AMPA receptor and the NK1 receptor. Conversely, signal transduction from MC3T3-E1 cells to DRG-derived neurons is suppressed by a P2X7 receptor antagonist. Our results suggest that these cells communicate with each other by exocytosis of glutamate, substance P in the efferent signal, and ATP in the afferent signal.

Background and purpose The sympathetic nervous system regulates bone remodelling, in part, through s2 -adrenoceptor signalling. However, the physiological role of α1 -adrenoceptor signalling in bone in vivo remains unclear. Therefore, to obtain a deeper understanding of bone remodelling by the sympathetic nervous system, we investigated the role of α1B -adrenoceptor signalling in bone metabolism. Experimental approach Prazosin, a nonspecific α1 -adrenoceptor antagonist, was administered for 2 weeks in C57BL6 mice, and efficacy was evaluated by bone microarchitecture using microcomputed tomography and determination of bone formation by fluorescent labelling of bone. We also compared the bone phenotype of α1B -adrenoceptor null mice (α1B (-/-) ) with that of wild-type littermates. Key results We demonstrated that the systemic administration of prazosin decreased bone formation. In addition, α1B -adrenoceptor-deficient mice had a lower bone mass due to decreased bone formation but did not exhibit any changes in bone-resorbing activity. Furthermore, stimulation with phenylephrine, a non-specific α1 -adrenoceptor agonist, increased the expression of the transcriptional factor CCAAT/enhancer-binding protein δ (Cebpd) in MC3T3-E1 osteoblastic cells. The overexpression of Cebpd induced cellular proliferation in MC3T3-E1 cells, whereas the silencing of Cebpd suppressed it. Conclusions and implications Taken together, these results suggested that α1B -adrenoceptor signalling is required for bone formation and regulated cellular proliferation through a mechanism relevant to the up-regulation of Cebpd in osteoblasts and, thus, provide new evidence for the physiological importance of α1B -adrenoceptor signalling in bone homeostasis.

The sympathetic nervous system is known to regulate osteoclast development. However, the involvement of α2-adrenergic receptors (α2-ARs) in osteoclastogenesis is not well understood. In the present study, their potential role in osteoclastogenesis was investigated. Guanabenz, clonidine and xylazine were used as agonists of α2-ARs, while yohimbine and idazoxan were employed as antagonists. Using RAW264.7 pre-osteoclast and primary bone marrow cells, the mRNA expression of the osteoclast-related genes nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), tartrate-resistant acid phosphatase (TRAP) and cathepsin K was evaluated following induction with receptor activator of nuclear factor κB ligand (RANKL). TRAP staining was also conducted to assess effects on osteoclastogenesis in mouse bone marrow cells in vitro. Administration of 5–20 µM guanabenz (P

An animal experiment clarified that insertion of an orthodontic apparatus activated the trigeminal neurons of the medulla oblongata. Orthodontic tooth movement is known to be associated with the sympathetic nervous system and controlled by the nucleus of the hypothalamus. However, the transmission of both has not been demonstrated in humans. The purpose of this study were to examine the activated cerebral areas using brain functional magnetic resonance imaging (MRI), when orthodontic tooth separators were inserted, and to confirm the possibility of the transmission route from the medulla oblongata to the hypothalamus. Two types of alternative orthodontic tooth separators (brass contact gauge and floss) were inserted into the right upper premolars of 10 healthy volunteers. Brain functional T2*-weighted images and anatomical T1-weighted images were taken. The blood oxygenation level dependent (BOLD) signals following insertion of a brass contact gauge and floss significantly increased in the somatosensory association cortex and hypothalamic area. Our findings suggest the possibility of a transmission route from the medulla oblongata to the hypothalamus. Identifying the nerve pathways involved in tooth movement could lead to better targets for pain relief. Non-steroidal anti-inflammatory drugs cannot be used to relieve orthodontic pain because they impair the processes involved in tooth movement. Yoshiko Ariji of Japan’s Aichi-Gakuin University School of Dentistry, Nagoya, and colleagues used functional MRI scans of ten healthy adult volunteers to identify the parts of the brain that become active when separators are briefly inserted between pre-molar teeth. They found separator insertion led to a significant rise in the activity of the hypothalamus and the part of the brain’s cerebral cortex associated with touch and proprioception. Together with the results of previous studies in mice, the results suggest a nerve pathway that could be targeted to alleviate pain from orthodontic procedures without negatively impacting tooth movement.

Background Circadian rhythms have been identified in bone metabolism. However, the underlying molecular mechanisms are poorly understood. It was recently reported that circadian rhythms are regulated by clock genes. The main circadian rhythm is regulated by endogenous circadian clocks in the suprachiasmatic nuclei (SCN). Highlight Recently, we reported that output signals from the SCN are transmitted from the master circadian rhythm to peripheral osteoblasts and osteoclasts through β-adrenergic signaling and/or glucocorticoids. Treatment with the β-agonist isoprenaline and the synthetic glucocorticoid dexamethasone synchronized the clock genes and osteoblast-related genes in cultured human osteoblasts. In addition, treatment with dexamethasone, but not isoprenaline, synchronized clock genes and osteoclast-related genes in cultured mouse osteoclasts. A chromatin immune-precipitation (ChIP) assay showed the interaction between brain and muscle Arnt-like protein (BMAL) and the promoter regions of osteoclast-related genes, such as cathepsin K ( Ctsk ) and nuclear factor of activated T-cells cytoplasmic 1 ( Nfatc 1). To examine whether endogenous glucocorticoids influence the circadian rhythms of bone metabolism, mice were adrenalectomized (ADX) or sham operated. These mice were maintained under a 12:12 h light:dark cycle for two weeks, and mRNA was collected from the femur every 4 h for a 24-h period. In the sham-operated mice, the expression of Ctsk and Nfatc 1 showed circadian rhythms. However, in the ADX mice, the circadian rhythms of Ctsk and Nfatc 1 expression disappeared. Conclusion These findings suggest that β-adrenergic signaling and/or glucocorticoids mediate circadian timing in peripheral osteoblasts and osteoclasts, and that BMAL contribute to the circadian rhythm of osteoclasts.

Gangliosides are widely expressed in almost all tissues and cells and are also considered to be essential in the development and maintenance of various organs and tissues. However, little is known about their roles in bone metabolism. In this study, we investigated the effects of genetic deletion of ganglioside D3 (GD3) synthase, which is responsible for the generation of all b-series gangliosides, on bone metabolism. Although b-series gangliosides were not expressed in osteoblasts, these gangliosides were expressed in pre-osteoclasts. However, the expression of these gangliosides was decreased after induction of osteoclastogenesis by receptor activator of nuclear factor kappa-B ligand (RANKL). Three-dimensional micro-computed tomography (3D-&mu, CT) analysis revealed that femoral cancellous bone mass in GD3 synthase-knockout (GD3S KO) mice was higher than that in wild type (WT) mice at the age of 40 weeks, although there were no differences in that between GD3S KO and WT mice at 15 weeks old. Whereas bone formation parameters (osteoblast numbers/bone surface and osteoblast surface/bone surface) in GD3S KO mice did not differ from WT mice, bone resorption parameters (osteoclast numbers/bone surface and osteoclast surface/bone surface) in GD3S KO mice became significantly lower than those in WT mice at 40 weeks of age. Collectively, this study demonstrates that deletion of GD3 synthase attenuates bone loss that emerges with aging.

Experimental tooth movement (ETM) changes the distribution of sensory nerve fibers in periodontal ligament and the bone architecture through the stimulation of bone remodeling. As the sympathetic nervous system is involved in bone remodeling, we examined whether ETM is controlled by sympathetic signaling or not. In male mice, elastic rubber was inserted between upper left first molar (M1) and second molar (M2) for 3 or 5 days. Nerve fibers immunoreactive for not only sensory neuromarkers, such as calcitonin gene-related peptide (CGRP), but also sympathetic neuromarkers, such as tyrosine hydroxylase (TH) and neuropeptide Y (NPY) were increased in the periodontal ligament during ETM. To elucidate the effect of the sympathetic signal mediated by ETM, mice were intraperitoneally injected with a β-antagonist, propranolol (PRO: 20 μg/g/day), or a β-agonist, isoproterenol (ISO: 5 μg/g/day) from 7 days before ETM. PRO treatment suppressed the amount of tooth movement by 12.9% in 3-day ETM and by 32.2% in 5-day ETM compared with vehicle treatment. On the other hand, ISO treatment increased it. Furthermore, ETM remarkably increased the osteoclast number on the bone surface (alveolar socket) (Oc.N/BS) in all drug treatments. PRO treatment suppressed Oc.N/BS by 39.4% in 3-day ETM, while ISO treatment increased it by 32.1% in 3-day ETM compared with vehicle treatment. Chemical sympathectomy using 6-hydroxydopamine (6-OHDA: 250 μg/g) showed results similar to those for PRO treatment in terms of both the amount of tooth movement and osteoclast parameters. Our data showed that blockade of sympathetic signaling inhibited the tooth movement and osteoclast increase induced by ETM, and stimulation of sympathetic signaling accelerated these responses. These data suggest that the mechano-adaptive response induced by ETM is controlled by sympathetic signaling through osteoclast activation.

Summary Bone is innervated by sympathetic and sensory neurons, which play important roles in bone remodeling. Direct neuro-osteogenic cross-talk has been demonstrated using an in vitro co-culture model comprising osteoblastic or osteoclastic cells, and neurite-spouting mouse superior cervical ganglia, suggesting that these cells are directly regulated by sympathetic neurons. The increase in sympathetic nervous activity causes bone loss through increased bone resorption and decreased bone formation, associated with β2-adrenergic activity toward both osteoblastic and osteoclastic cells. The increased bone resorption is based on the stimulation of both osteoclastogenesis and osteoclastic activity. These studies suggest β-blockers to be effective against osteoporosis, in which case there is increased sympathetic activity. Epidemiological studies have demonstrated high blood pressure to be associated with increased bone loss and β-blockers to be potential candidates for drugs to treat osteoporosis and fractures. In animal experiments, a lower dose of β-blocker also improved bone mass and biomechanical fragility in a hypertensive model. Although the interplay between sympathetic and sensory neurons is poorly understood, interestingly, sensory denervated rats have been demonstrated to show similar bone metabolism to rats with hyperactivity of sympathetic tone. This review summarized both in vitro and in vivo evidence implicating sympathetic neuronal activity in bone resorption.

The sympathetic nervous system regulates bone remodelling, in part, through ß2 -adrenoceptor signalling. However, the physiological role of α1 -adrenoceptor signalling in bone in vivo remains unclear. Therefore, to obtain a deeper understanding of bone remodelling by the sympathetic nervous system, we investigated the role of α1B -adrenoceptor signalling in bone metabolism.Prazosin, a nonspecific α1 -adrenoceptor antagonist, was administered for 2 weeks in C57BL6 mice, and efficacy was evaluated by bone microarchitecture using microcomputed tomography and determination of bone formation by fluorescent labelling of bone. We also compared the bone phenotype of α1B -adrenoceptor null mice (α1B (-/-) ) with that of wild-type littermates.We demonstrated that the systemic administration of prazosin decreased bone formation. In addition, α1B -adrenoceptor-deficient mice had a lower bone mass due to decreased bone formation but did not exhibit any changes in bone-resorbing activity. Furthermore, stimulation with phenylephrine, a non-specific α1 -adrenoceptor agonist, increased the expression of the transcriptional factor CCAAT/enhancer-binding protein δ (Cebpd) in MC3T3-E1 osteoblastic cells. The overexpression of Cebpd induced cellular proliferation in MC3T3-E1 cells, whereas the silencing of Cebpd suppressed it.Taken together, these results suggested that α1B -adrenoceptor signalling is required for bone formation and regulated cellular proliferation through a mechanism relevant to the up-regulation of Cebpd in osteoblasts and, thus, provide new evidence for the physiological importance of α1B -adrenoceptor signalling in bone homeostasis.

Bone remodelling, the mechanism by which vertebrates regulate bone mass, comprises two phases, namely resorption by osteoclasts and formation by osteoblasts; osteoblasts are multifunctional cells also controlling osteoclast differentiation. Sympathetic signalling via beta2-adrenergic receptors (Adrb2) present on osteoblasts controls bone formation downstream of leptin. Here we show, by analysing Adrb2-deficient mice, that the sympathetic nervous system favours bone resorption by increasing expression in osteoblast progenitor cells of the osteoclast differentiation factor Rankl. This sympathetic function requires phosphorylation (by protein kinase A) of ATF4, a cell-specific CREB-related transcription factor essential for osteoblast differentiation and function. That bone resorption cannot increase in gonadectomized Adrb2-deficient mice highlights the biological importance of this regulation, but also contrasts sharply with the increase in bone resorption characterizing another hypogonadic mouse with low sympathetic tone, the ob/ob mouse. This discrepancy is explained, in part, by the fact that CART ('cocaine amphetamine regulated transcript'), a neuropeptide whose expression is controlled by leptin and nearly abolished in ob/ob mice, inhibits bone resorption by modulating Rankl expression. Our study establishes that leptin-regulated neural pathways control both aspects of bone remodelling, and demonstrates that integrity of sympathetic signalling is necessary for the increase in bone resorption caused by gonadal failure.

Circadian rhythms are prevalent in bone metabolism. However, the molecular mechanisms involved are poorly understood. Recently, we suggested that output signals from the suprachiasmatic nucleus (SCN) are transmitted from the master circadian rhythm to peripheral osteoblasts through β-adrenergic and glucocorticoid signaling. In this study, we examined how the master circadian rhythm is transmitted to peripheral osteoclasts and the role of clock gene in osteoclast. Mice were maintained under 12-hour light/dark periods and sacrificed at Zeitgeber times 0, 4, 8, 12, 16 and 20. mRNA was extracted from femur (cancellous bone) and analyzed for the expression of osteoclast-related genes and clock genes. Osteoclast-related genes such as cathepsin K (CTSK) and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) showed circadian rhythmicity like clock genes such as period 1 (PER1), PER2 and brain and muscle Arnt-like protein 1 (BMAL1). In an in vitro study, not β-agonist but glucocorticoid treatment remarkably synchronized clock and osteoclast-related genes in cultured osteoclasts. Chromatin immunoprecipitation (ChIP) assay showed the interaction between BMAL1 proteins and promoter region of CTSK and NFATc1. To examine whether endogenous glucocorticoids influence the osteoclast circadian rhythms, mice were adrenalectomized (ADX) and maintained under 12-hour light/dark periods at least two weeks before glucocorticoid injection. A glucocorticoid injection restarted the circadian expression of CTSK and NFATc1 in ADX mice. These results suggest that glucocorticoids mediate circadian timing to peripheral osteoclasts and osteoclast clock contributes to the circadian expression of osteoclast-related genes such as CTSK and NFATc1.

Sympathetic signaling regulates bone resorption through receptor activator of nuclear factor-κB ligand (RANKL) expression via the β-adrenergic receptor (β-AR) on osteoblasts. Reactive oxygen species (ROS) are known as one type of osteoclast regulatory molecule. Here we show that an antioxidant, α-lipoic acid (α-LA), treatment prevent the β-adrenergic signaling-induced bone loss by suppressing osteoclastogenesis, and sympathetic signaling directly regulates osteoclastogenesis through β2-AR expressed on osteoclasts via intracellular ROS generation. In an in vitro study, the β-AR agonist isoprenaline increased intracellular ROS generation in osteoclasts prepared from bone marrow macrophages (BBMs) and RAW 264.7 cells. Isoprenaline enhanced osteoclastogenesis through β2-AR expressed on BMMs and RAW 264.7 cells. The antioxidant α-LA inhibited isoprenaline-enhanced osteoclastogenesis. Isoprenaline increased the expression of osteoclast-related genes such as nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1, tartrate-resistant acid phosphatase, and cathepsin K on osteoclasts. α-LA also inhibited isoprenaline-induced increases of these gene expressions. These in vitro results led to the hypothesis that β-adrenergic signaling directly stimulates osteoclastogenesis via ROS generation. In an in vivo study, isoprenaline treatment alone caused oxidative damage in local bone and reduced bone mass because of an increase in bone resorption, and, in α-LA-treated mice, isoprenaline did not increase tibial osteoclast number even though the RANKL-to-osteoprotegerin ratio increased. These in vitro and in vivo results indicate that β-adrenergic signaling, at least in part, directly stimulates osteoclastogenesis through β2-AR on osteoclasts via ROS generation.

Loss of mechanical stress or unloading causes disuse osteoporosis that leads to fractures and deteriorates body function and affects mortality rate in aged population. This bone loss is due to reduction in osteoblastic bone formation and increase in osteoclastic bone resorption. MuRF1 is a muscle RING finger protein which is involved in muscle wasting and its expression is enhanced in the muscle of mice subjected to disuse condition such as hind limb unloading (HU). However, whether MuRF1 is involved in bone loss due to unloading is not known. We therefore examined the effects of MuRF1 deficiency on unloading-induced bone loss. We conducted hind limb unloading of MuRF1 KO mice and wild-type control mice. Unloading induced about 60% reduction in cancellous bone volume (BV/TV) in WT mice. In contrast, MuRF1 deficiency suppressed unloading-induced cancellous bone loss. The cortical bone mass was also reduced by unloading in WT mice. In contrast, MuRF1 deficiency suppressed this reduction in cortical bone mass. To understand whether the effects of MuRF1 deficiency suppress bone loss is on the side of bone formation or bone resorption, histomorphometry was conducted. Unloading reduced bone osteoblastic formation rate (BFR) in WT. In contrast, MuRF1 deficiency suppressed this reduction. Regarding bone resorption, unloading increased osteoclast number in WT. In contrast, MURF1 deficiency suppressed this osteoclast increase. These data indicated that the ring finger protein, MURF1 is involved in disuse-induced bone loss in both of the two major bone remodeling activities, osteoblastic bone formation and osteoclastic bone resorption.

The sympathetic nervous system regulates bone remodeling through the β-adrenergic receptor (β-AR). However, the systemic roles of adrenergic actions on bone remodeling through the β-AR are largely unknown. In this study, we examined the dose effect of continuous treatment with isoprenaline, a nonspecific β-AR agonist, on bone remodeling. Male C57BL/6J mice were intrasubcutaneously administrated with four different doses (5, 25, 50, or 100 μg/g daily) of isoprenaline or vehicle using an osmotic pump for 2 weeks. The region of high-turnover cancellous bone was analyzed by microcomputed tomography (μCT). Continuous isoprenaline treatment caused a ~35.7% decline in the femoral cancellous bone volume fraction (BV/TV) at all doses (5–100 μg/g daily). Furthermore, continuous isoprenaline treatment weakened the bone mechanical properties in the trunk of lumbar vertebra 4 (L4). These parameters did not show significant differences between doses. Histomorphometric analysis revealed that isoprenaline doses of 50 μg/g daily or less did not significantly inhibit bone formation parameters, such as bone formation rate (BFR) and mineral surface/bone surface (MS/BS). Only the highest dose (100 μg/g daily) of isoprenaline significantly inhibited BFR and MS/BS. On the other hand, osteoclast number/bone surface (Oc.N/BS) was enhanced approximately 2.4-fold and osteoclast surface/bone surface (Oc.S/BS) was increased 2.0-fold by all doses of continuous isoprenaline treatment. The osteoclast parameters plateaued at the lowest dose (5 μg/g daily) of continuous isoprenaline treatment. These results indicate that chronic stimulation of β-AR with low-dose agonist treatment induces bone loss mainly via enhanced bone resorption.

Exposure of astronauts in space to radiation during weightlessness may contribute to subsequent bone loss. Gamma irradiation of postpubertal mice rapidly increases the number of bone-resorbing osteoclasts and causes bone loss in cancellous tissue; similar changes occur in skeletal diseases associated with oxidative stress. Therefore, we hypothesized that increased oxidative stress mediates radiation-induced bone loss and that musculoskeletal disuse changes the sensitivity of cancellous tissue to radiation exposure. Musculoskeletal disuse by hindlimb unloading (1 or 2 wk) or total body gamma irradiation (1 or 2 Gy of 137Cs) of 4-mo-old, male C57BL/6 mice each decreased cancellous bone volume fraction in the proximal tibiae and lumbar vertebrae. The extent of radiation-induced acute cancellous bone loss in tibiae and lumbar vertebrae was similar in normally loaded and hindlimb-unloaded mice. Similarly, osteoclast surface in the tibiae increased 46% as a result of irradiation, 47% as a result of hindlimb unloading, and 64% as a result of irradiation + hindlimb unloading compared with normally loaded mice. Irradiation, but not hindlimb unloading, reduced viability and increased apoptosis of marrow cells and caused oxidative damage to lipids within mineralized tissue. Irradiation also stimulated generation of reactive oxygen species in marrow cells. Furthermore, injection of α-lipoic acid, an antioxidant, mitigated the acute bone loss caused by irradiation. Together, these results showed that disuse and gamma irradiation, alone or in combination, caused a similar degree of acute cancellous bone loss and shared a common cellular mechanism of increased bone resorption. Furthermore, irradiation, but not disuse, may increase the number of osteoclasts and the extent of acute bone loss via increased reactive oxygen species production and ensuing oxidative damage, implying different molecular mechanisms. The finding that α-lipoic acid protected cancellous tissue from the detrimental effects of irradiation has potential relevance to astronauts and radiotherapy patients.

Bone metabolism has recently been revealed to be under nerve regulation. In this study, the integrity of the sensory innervation contributing to bone metabolism was examined by capsaicin-induced sensory neuron lesions. Eight-week-old male Wistar strain rats in a modeling phase of skeletal growth were divided into four groups (8 rats per group) and treated with capsaicin at one of three different doses (37.5, 75, 150 mg/kg) or vehicle, subcutaneously. Five weeks later, high-dose (150 mg/kg) capsaicin treatment had reduced trabecular bone volume (BV/TV) due to increased trabecular separation (Tb.Sp) in the proximal tibia and the modification of mechanical properties such as strength, ductility, and toughness toward increasing bone fragility in the trunk of the sixth lumbar vertebrae (L6). Moderate-dose (75 mg/kg) capsaicin treatment had no significant effect on trabecular BV/TV or bone mechanical properties but increased Tb.Sp as seen high-dose capsaicin treatment. Bone histomorphometry showed osteoclast number (Oc.N/BS) and surface (Oc.S/BS) were increased in both the moderate-dose and high-dose capsaicin treatment groups. High-dose capsaicin significantly increased the level of tartrate-resistant acid phosphatase form 5b (TRAP 5b) in plasma, a systemic bone resorption marker, but had no influence on plasma osteocalcin concentration, a bone formation marker, suggesting that capsaicin-induced sensory nerve denervation increased bone resorption but had no influence on bone formation. Low-dose (37.5mg/kg) capsaicin had no influence on bone remodeling. These results suggest that sensory nerve innervation contributes to the maintenance of trabecular bone mass and its mechanical properties by inhibiting bone resorption.

Osteoporosis is one of the major health problems in our modern world. Especially, disuse (unloading) osteoporosis occurs commonly in bedridden patients, a population that is rapidly increasing due to aging-associated diseases. However, the mechanisms underlying such unloading-induced pathological bone loss have not yet been fully understood. Since sympathetic nervous system could control bone mass, we examined whether unloading-induced bone loss is controlled by sympathetic nervous tone. Treatment with beta-blocker, propranolol, suppressed the unloading-induced reduction in bone mass. Conversely, beta-agonist, isoproterenol, reduced bone mass in loaded mice, and under such conditions, unloading no longer further reduced bone mass. Analyses on the cellular bases indicated that unloading-induced reduction in the levels of osteoblastic cell activities, including mineral apposition rate, mineralizing surface, and bone formation rate, was suppressed by propranolol treatment and that isoproterenol-induced reduction in these levels of bone formation parameters was no longer suppressed by unloading. Unloading-induced reduction in the levels of mineralized nodule formation in bone marrow cell cultures was suppressed by propranolol treatment in vivo. In addition, loss of a half-dosage in the dopamine beta-hydroxylase gene suppressed the unloading-induced bone loss and reduction in mineralized nodule formation. Unloading-induced increase in the levels of osteoclastic activities such as osteoclast number and surface as well as urinary deoxypyridinoline was all suppressed by the treatment with propranolol. These observations indicated that sympathetic nervous tone mediates unloading-induced bone loss through suppression of bone formation by osteoblasts and enhancement of resorption by osteoclasts.