Hisataka Kondo

Glucocorticoids mediate circadian timing in peripheral osteoclasts resulting in the circadian expression rhythm of osteoclast-related genes

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.

Effects of capsaicin-induced sensory denervation on bone metabolism in adult rats

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.

β-Adrenergic signaling stimulates osteoclastogenesis via reactive oxygen species

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.

Experimental tooth movement-induced osteoclast activation is regulated by sympathetic signaling.

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.

α1B‐Adrenoceptor signalling regulates bone formation through the up‐regulation of CCAAT/enhancer‐binding protein δ expression in osteoblasts

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.

Bidirectional communication between sensory neurons and osteoblasts in an in vitro coculture system

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.

Conditioned medium from rat dental pulp reduces the number of osteoclasts via attenuation of adhesiveness in osteoclast precursors

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.

Orthodontic tooth separation activates the hypothalamic area in the human brain

ObjectivesAn 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.MethodsTwo 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.ResultsThe 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.ConclusionOur findings suggest the possibility of a transmission route from the medulla oblongata to the hypothalamus.Orthodontics: The nerve pathways of tooth movementIdentifying 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.

Suppression of osteoclastogenesis via α2‑adrenergic receptors

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<0.01, for RANKL-only treatment), 20 µM clonidine (P<0.05, for RANKL-only treatment) and 20 µM xylazine (P<0.05, for RANKL-only treatment) attenuated RANKL-induced upregulation of NFATc1, TRAP and cathepsin K mRNA. Furthermore, the reductions in these mRNAs by 10 µM guanabenz and 20 µM clonidine in the presence of RANKL were attenuated by 20 µM yohimbine or idazoxan (P<0.05). The administration of 5–20 µM guanabenz (P<0.01, for RANKL-only treatment) and 10–20 µM clonidine (P<0.05, for RANKL-only treatment) also decreased the number of TRAP-positive multi-nucleated osteoclasts. Collectively, the present study demonstrates that α2-ARs may be involved in the regulation of osteoclastogenesis.

Experimental tooth movement-induced osteoclast activation is regulated by sympathetic signaling

cells, and was remarkably down regulated when the cells were treated with a GJ blocker. Additionally, we examined GJs under hypoxic stress. The fluorescence recovery and expression levels of Cx43 decreased time-dependently under the hypoxic condition. Exposure to GJ inhibitor or hypoxia increased RANKL expression, and decreased OPG expression. This study shows that GJIC is responsible for hPDL cells and that its activity is reduced under hypoxia. This is consistent with the possible role of hPDL cells in regulating the biochemical reactions in response to changes in the hypoxic environment.