Attenuation of Bone Formation through a Decrease in Osteoblasts in Mutant Mice Lacking the GM2/GD2 Synthase Gene.

The ganglioside GD1a has been reported to promote the differentiation of mesenchymal stem cells to osteoblasts in cell culture systems. However, the involvement of gangliosides, including GD1a, in bone formation in vivo remains unknown; therefore, we herein investigated their roles in GM2/GD2 synthase-knockout (GM2/GD2S KO) mice without GD1a. The femoral cancellous bone mass was analyzed using three-dimensional micro-computed tomography. A histomorphometric analysis of bone using hematoxylin and eosin (HE) and tartrate-resistant acid phosphatase was performed to examine bone formation and resorption, respectively. Calcein double labeling was also conducted to evaluate bone formation. Although no significant differences were observed in bone mass or resorption between GM2/GD2S KO mice and wild-type (WT) mice, analyses of the parameters of bone formation using HE staining and calcein double labeling revealed less bone formation in GM2/GD2S KO mice than in WT mice. These results suggest that gangliosides play roles in bone formation.

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.

Suppression of alveolar bone resorption by salubrinal in a mouse model of periodontal disease.

AIMS: The relationship between stress to endoplasmic reticulum (ER) and periodontitis has been known, and ER stress induced by Porphyromonas gingivalis results in the loss of alveolar bone. Salubrinal is a small synthetic compound and attenuates ER stress through inhibition of de-phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). In this study, we examined whether salubrinal attenuates periodontitis in a mouse model of experimental periodontal disease. MATERIALS AND METHODS: We evaluated loss of alveolar bone and attachment levels in periodontium using micro-computed tomography (µCT) and hematoxylin-eosin (HE) staining, respectively. Furthermore, we measured osteoclast numbers using tartrate-resistant acid phosphatase (TRAP) staining and osteoblast numbers using HE staining for bone resorption and for bone formation, respectively. To examine the inhibitory effects of salubrinal against pro-inflammatory cytokines, we measured TNF-α and IL1-ß score in periodontium using immunohistostaining. KEY FINDINGS: The results revealed that salubrinal suppressed loss of alveolar bone and attachment levels in periodontium induced by periodontitis. It decreased osteoclast numbers and increased osteoblasts. It also suppressed the expression levels of TNF-α in periodontium. SIGNIFICANCE: These results show that salubrinal alleviates periodontitis through suppression of alveolar bone resorption and the pro-inflammatory cytokine, and promotion of the bone formation. Since salubrinal has been shown to have these beneficial effects for periodontal disease, it may provide a novel therapeutic possibility for the disease.

Guanabenz inhibits alveolar bone resorption in a rat model of periodontitis.

Increase of sympathetic activity has been known to exacerbate osteoporosis through promotion of bone resorption. However, it is largely unknown about involvement of sympathetic activity in exacerbation of periodontitis. In this study, we investigated whether α2-adrenergic receptor (α2-AR) agonist guanabenz which decreases sympathetic activity, attenuates alveolar bone resorption in rats having high sympathetic activity with periodontitis. Volumes of residual alveolar bone and attachment levels in periodontium were examined using micro-computed tomography and hematoxylin-eosin staining, respectively. Furthermore, osteoclast numbers per bone surface and osteoclast surface per bone surface were measured using tartrate-resistant acid phosphatase staining. To examine the suppressive effects of guanabenz on pro-inflammatory cytokines, expression levels of tyrosine hydroxylase (TH), TNF-α, IL1-ß, and IL-6 in periodontium were measured using immunohistostaining. Administration of guanabenz attenuated loss of alveolar bone and attachment levels in rats having high sympathetic activity. Furthermore, its administration suppressed osteoclast numbers in rats having high sympathetic activity. TH, TNF-α, IL-1ß, and IL-6 positive cells in periodontium in rats treated with guanabenz for 12 weeks, were lower than those in control rats having high sympathetic activity. This study demonstrated administration of α2-AR agonist guanabenz attenuates alveolar bone resorption through decrease of sympathetic activity in rats.

Effects of a ß2-adrenergic receptor blocker on experimental periodontitis in spontaneously hypertensive rats.

AIMS: Recent studies have reported a relationship between periodontal disease and hypertension, and previous evidence suggests that the sympathetic nervous system plays an important role in the control of bone metabolism. This study sought to evaluate the effect of the beta-2 adrenergic receptor (ß2-AR) blocker butoxamine on experimental periodontitis in a rat model. MATERIALS AND METHODS: Wistar-Kyoto and spontaneously hypertensive rats (n = 6 per group) were orally administered butoxamine 1 mg/kg/day and experimental periodontitis was induced by applying an orthodontic ligature wire. The rats were sacrificed after 4 weeks and the residual alveolar bone was measured using micro-computed tomography (micro-CT) imaging analysis software for histological analysis. KEY FINDINGS: Micro-CT imaging analysis showed a higher ratio of residual alveolar bone, BV/TV, and Tb.N in both Wistar-Kyoto and spontaneously hypertensive rats treated with butoxamine compared with the corresponding control rats. In histological analysis, compared with the Wistar-Kyoto and spontaneously hypertensive rat control groups, the corresponding butoxamine-treated groups showed a lower ratio of attachment level, lower values of osteoclast number and surface. SIGNIFICANCE: ß2-AR blockers maintained the alveolar bone mass and attachment level by suppressing osteoclast activity. Thus, ß2-AR blockers may be effective in preventing periodontitis.

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.

α1B-Adrenergic receptor signaling controls circadian expression of Tnfrsf11b by regulating clock genes in osteoblasts.

Circadian clocks are endogenous and biological oscillations that occur with a period of <24 h. In mammals, the central circadian pacemaker is localized in the suprachiasmatic nucleus (SCN) and is linked to peripheral tissues through neural and hormonal signals. In the present study, we investigated the physiological function of the molecular clock on bone remodeling. The results of loss-of-function and gain-of-function experiments both indicated that the rhythmic expression of Tnfrsf11b, which encodes osteoprotegerin (OPG), was regulated by Bmal1 in MC3T3-E1 cells. We also showed that REV-ERBα negatively regulated Tnfrsf11b as well as Bmal1 in MC3T3-E1 cells. We systematically investigated the relationship between the sympathetic nervous system and the circadian clock in osteoblasts. The administration of phenylephrine, a nonspecific α1-adrenergic receptor (AR) agonist, stimulated the expression of Tnfrsf11b, whereas the genetic ablation of α1B-AR signaling led to the alteration of Tnfrsf11b expression concomitant with Bmal1 and Per2 in bone. Thus, this study demonstrated that the circadian regulation of Tnfrsf11b was regulated by the clock genes encoding REV-ERBα (Nr1d1) and Bmal1 (Bmal1, also known as Arntl), which are components of the core loop of the circadian clock in osteoblasts.

Modulation of osteoblast differentiation and bone mass by 5-HT2A receptor signaling in mice.

Recent studies reported that serotonin (5-hydroxytryptamine, 5-HT) may be an endogenous paracrine and/or autocrine factor that is used for intercellular communication in bone cells and between multiple organs regulating bone homeostasis. In the present study, we showed that the administration of MDL11939, a selective 5-HT2A receptor antagonist, reduced bone mass in mice. The loss of bone mass in MDL11939-treated mice was associated with impaired bone formation in vivo, as demonstrated by the lower expression of osterix (Osx) and osteocalcin than that in vehicle-treated mice. On the other hand, no significant differences were observed in osteoclast numbers between MDL11939- and vehicle-treated mice. The pharmacological blockade of 5-HT2A receptor signaling significantly decreased alkaline phosphatase activity in osteoblastic cells. In addition, the knockdown of the 5-HT2A receptor by a siRNA treatment decreased Osx, but not Runx2 gene expression in MC3T3-E1 cells. These results suggest that 5-HT2A receptor signaling mediated bone mass by regulating osteoblast differentiation.

Shear stress-induced Ca(2+) elevation is mediated by autocrine-acting glutamate in osteoblastic MC3T3-E1 cells.

Mechanical loading is an important regulatory factor in bone homeostasis. Neurotransmitters, such as glutamate and ATP, are known to be released from osteoblasts, but their roles have been less studied. In this study, we investigated the role of transmitter release in mechanotransduction. To identify from where transmitters were released, focal fluid flow was applied to a single cell of MC3T3-E1, mouse calvaria-derived osteoblastic cell line, by using a glass micropipette. Intracellular Ca(2+) elevation induced by the focal shear stress was eliminated by either GdCl3, a mechanosensing channel inhibitor, or removal of extracellular Ca(2+). On the other hand, the focal shear stress-induced Ca(2+) elevation was also significantly suppressed by inositol triphosphate receptor antagonist or vesicular release inhibitors. These results suggest that not only mechanosensitive channel-mediated Ca(2+) influx but also some autocrine transmitters are involved in mechanotransduction. Additionally, glutamate receptor antagonists, but not ATP receptor antagonist, suppressed most of the focal shear stress-induced Ca(2+) elevation. Therefore, it is suggested that glutamate is released from osteoblasts following the activation of mechanosensitive Ca(2+) channels and acts in an autocrine manner. The glutamate release may have a significant role in the initial event of mechanotransduction in bone tissue.