Porphyromonas gingivalis Fimbriae Induce Osteoclastogenesis via Toll-like Receptors in RAW264 Cells.

The effect of Mfa1 fimbriae of Porphyromonas gingivalis on the progression of bone resorption remains unclear, especially compared with another fimbriae, FimA. We investigated the effect of Mfa1 on osteoclastogenesis together with FimA. We also investigated the role of Toll-like receptors (TLRs) in Mfa1 recognition during osteoclast differentiation. Receptor activator of nuclear factor κß ligand (RANKL)-prestimulated RAW264 cells were used to examine the effects of purified Mfa1 fimbriae. The number of osteoclasts was examined by tartrate-resistant acid phosphate (TRAP) staining, osteoclast activation was investigated by bone resorption assays, and gene expression of differentiation markers was examined by quantitative real-time PCR. Transfection of Tlr2 and Tlr4 siRNAs into RAW264 cells was also employed and their role in Mfa1 recognition was investigated. Mfa1 effectively induced the formation of TRAP-positive multinucleated cells and activated osteoclasts. Mfa1 also increased gene expression of Acp5, Mmp9, and Ctsk in RANKL-prestimulated RAW264 cells compared with the control. The osteoclastogenesis induced by Mfa1 was significantly decreased in cells transfected with Tlr2 or Tlr4 siRNAs compared with control siRNA. Our results revealed the role of Mfa1 fimbriae in osteoclastogenesis that may contribute to the partial elucidation of the mechanisms of periodontal disease progression and the development of new therapeutic strategies.

Orthodontic tooth movement-activated sensory neurons contribute to enhancing osteoclast activity and tooth movement through sympathetic nervous signalling.

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 ß-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.

Contribution of Glucosylceramide Synthase to the Proliferation of Mouse Osteoblasts.

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.

Co-cultured spheroids of human periodontal ligament mesenchymal stem cells and vascular endothelial cells enhance periodontal tissue regeneration.

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.

Suppression of tooth movement-induced sclerostin expression using ß-adrenergic receptor blockers.

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 ß-adrenergic receptor (ß-AR) blockers atenolol (ß1-AR blocker), butoxamine (ß2-AR blocker) and propranolol (non-selective ß-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 ß-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: ß-AR blockers decreased tooth movement and downregulated SOST in osteocytes, accompanied by increasing alveolar bone resorption.

Deletion of Gb3 Synthase in Mice Resulted in the Attenuation of Bone Formation via Decrease in Osteoblasts.

Glycosphingolipids are known to play a role in developing and maintaining the integrity of various organs and tissues. Among glycosphingolipids, there are several reports on the involvement of gangliosides in bone metabolism. However, there have been no reports on the presence or absence of expression of globo-series glycosphingolipids in osteoblasts and osteoclasts, and the involvement of their glycosphingolipids in bone metabolism. In the present study, we investigated the presence or absence of globo-series glycosphingolipids such as Gb3 (globotriaosylceramide), Gb4 (globoside), and Gb5 (galactosyl globoside) in osteoblasts and osteoclasts, and the effects of genetic deletion of Gb3 synthase, which initiates the synthesis of globo-series glycosphingolipids on bone metabolism. Among Gb3, Gb4, and Gb5, only Gb4 was expressed in osteoblasts. However, these glycosphingolipids were not expressed in pre-osteoclasts and osteoclasts. Three-dimensional micro-computed tomography (3D-µCT) analysis revealed that femoral cancellous bone mass in Gb3 synthase-knockout (Gb3S KO) mice was lower than that in wild type (WT) mice. Calcein double labeling also revealed that bone formation in Gb3S KO mice was significantly lower than that in WT mice. Consistent with these results, the deficiency of Gb3 synthase in mice decreased the number of osteoblasts on the bone surface, and suppressed mRNA levels of osteogenic differentiation markers. On the other hand, osteoclast numbers on the bone surface and mRNA levels of osteoclast differentiation markers in Gb3S KO mice did not differ from WT mice. This study demonstrated that deletion of Gb3 synthase in mice decreases bone mass via attenuation of bone formation.

Deficiency of GD3 Synthase in Mice Resulting in the Attenuation of Bone Loss with Aging.

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-µCT) 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.

Systemic administration of low-dose naltrexone increases bone mass due to blockade of opioid growth factor receptor signaling in mice osteoblasts.

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.

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.

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.

Orthodontic tooth separation activates the hypothalamic area in the human brain.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Our findings suggest the possibility of a transmission route from the medulla oblongata to the hypothalamus.

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.

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

BACKGROUND AND PURPOSE: 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. 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.

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 ß-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 ß-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.

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 ß-antagonist, propranolol (PRO: 20 µg/g/day), or a ß-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.

ß-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 ß-adrenergic receptor (ß-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 ß-adrenergic signaling-induced bone loss by suppressing osteoclastogenesis, and sympathetic signaling directly regulates osteoclastogenesis through ß2-AR expressed on osteoclasts via intracellular ROS generation. In an in vitro study, the ß-AR agonist isoprenaline increased intracellular ROS generation in osteoclasts prepared from bone marrow macrophages (BBMs) and RAW 264.7 cells. Isoprenaline enhanced osteoclastogenesis through ß2-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 ß-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 ß-adrenergic signaling, at least in part, directly stimulates osteoclastogenesis through ß2-AR on osteoclasts via ROS generation.

Comparison of ß-adrenergic and glucocorticoid signaling on clock gene and osteoblast-related gene expressions in human osteoblast.

Most living organisms exhibit circadian rhythms that are generated by endogenous circadian clocks, the master one being present in the suprachiasmatic nuclei (SCN). Output signals from the SCN are believed to transmit standard circadian time to peripheral tissue through sympathetic nervous system and humoral routes. Therefore, the authors examined the expression of clock genes following treatment with the ß-adrenergic receptor agonist, isoprenaline, or the synthetic glucocorticoid, dexamethasone, in cultured human osteoblast SaM-1 cells. Cells were treated with 10(-6) M isoprenaline or 10(-7) M dexamethasone for 2 h and gene expressions were determined using real-time polymerase chain reaction (PCR) analysis. Treatment with isoprenaline or dexamethasone induced the circadian expression of clock genes human period 1 (hPer1), hPer2, hPer3, and human brain and muscle Arnt-like protein 1 (hBMAL1). Isoprenaline or dexamethasone treatment immediately increased hPer1 and hPer2 and caused circadian oscillation of hPer1 and hPer2 with three peaks within 48 h. hPer3 expression had one peak after isoprenaline or dexamethasone treatment. hBMAL expression had two peaks after isoprenaline or dexamethasone treatment, the temporal pattern being in antiphase to that of the other clock genes. Dexamethasone treatment delayed the oscillation of all clock genes for 2-6 h compared with isoprenaline treatment. The authors also examined the expression of osteoblast-related genes hα-1 type I collagen (hCol1a1), halkaline phosphatase (hALP), and hosteocalcin (hOC). Isoprenaline induced oscillation of hCol1a1, but not hALP and hOC. On the other hand, dexamethasone induced oscillation of hCol1a1 and hALP, but not hOC. Isoprenaline up-regulated hCol1a1 expression, but dexamethasone down-regulated hCol1a1 and hALP expression in the first phase.

MURF1 deficiency suppresses unloading-induced effects on osteoblasts and osteoclasts to lead to bone loss.

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.

Continuous treatment with a low-dose ß-agonist reduces bone mass by increasing bone resorption without suppressing bone formation.

The sympathetic nervous system regulates bone remodeling through the ß-adrenergic receptor (ß-AR). However, the systemic roles of adrenergic actions on bone remodeling through the ß-AR are largely unknown. In this study, we examined the dose effect of continuous treatment with isoprenaline, a nonspecific ß-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 ß-AR with low-dose agonist treatment induces bone loss mainly via enhanced bone resorption.