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.

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.

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.

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.

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.

ß-adrenergic receptor signaling regulates Ptgs2 by driving circadian gene expression in osteoblasts.

The sympathetic nervous system modulates bone remodeling and mediates the expression of core clock genes in part through the ß-adrenergic receptor (ß-AR) in osteoblasts. In this study, we show that in MC3T3-E1 osteoblastic cells that isoproterenol (Iso), a non-selective ß-AR agonist, upregulated the transcriptional factor Nfil3, and induced rhythmic mRNA expression of prostaglandin-endoperoxide synthase 2 (Ptgs2, also known as Cox2). The rhythmic effects of Iso on Ptgs2 expression were mediated by interplay between the Per2 and Bmal1 clock genes in osteoblasts. In addition, Ptgs2 was significantly decreased in bone after continuous Iso treatment. Overexpression of Nfil3 decreased Ptgs2 expression in MC3T3-E1 cells. Knockdown of Nfil3 upregulated the expression of Ptgs2 in MC3TC-E1 cells, indicating that Nfil3 negatively regulated Ptgs2 in osteoblasts. Furthermore, Iso acutely induced the expression Nfil3 and increased the binding of Nfil3 to the Ptgs2 promoter in MC3T3-E1 cells. These results suggest that Iso-mediated induction of Nfil3 in osteoblasts regulates the expression of Ptgs2 by driving the expression of circadian clock genes. These findings provide new evidence for a physiological role of circadian clockwork in bone metabolism.

α1-adrenergic receptor signaling in osteoblasts regulates clock genes and bone morphogenetic protein 4 expression through up-regulation of the transcriptional factor nuclear factor IL-3 (Nfil3)/E4 promoter-binding protein 4 (E4BP4).

Several studies have demonstrated that the α1-adrenergic receptor (AR) plays an important role in regulating cell growth and function in osteoblasts. However, the physiological role of α1-AR signaling in bone metabolism is largely unknown. In this study, the stimulation of phenylephrine (PHE), a nonspecific α1-AR agonist, increased the transcriptional factor Nfil3/E4BP4 and led to the rhythmic expression of bone morphogenetic protein 4 (Bmp4) in MC3T3-E1 osteoblastic cells. We also showed that Bmp4 mRNA expression peaked in bone near zeitgeber time 8 in a 24-h rhythm. Furthermore, the expression of Nfil3 and Bmp4 displayed a circadian pattern with opposing phases, which suggested that Nfil3 repressed the expression of the Bmp4 gene during a circadian cycle. On a molecular level, both loss-of-function and gain-of-function experiments demonstrated that Nfil3/E4BP4 negatively regulated Bmp4 expression in osteoblasts. Furthermore, the systemic administration of PHE increased the expression of Nfil3 mRNA in bone, whereas it decreased that of Bmp4 mRNA. The expression of Bmp4 mRNA was decreased significantly by exposure to PHE, and this was concomitant with the increase in Nfil3 binding to the D-box-containing Bmp4 promoter region in MC3T3-E1 cells, which indicates that the expression of Nfil3 by α1-AR signaling can bind directly to the Bmp4 promoter and inhibit Bmp4 expression in osteoblasts. Our results suggest that α1-AR signaling regulates clock genes and Bmp4 expression in osteoblasts. Moreover, α1-AR signaling negatively regulated Bmp4 expression by up-regulating the transcriptional factor Nfil3/E4BP4 in osteoblasts.

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.

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.

Store-operated calcium entry induced by activation of Gq-coupled alpha1B adrenergic receptor in human osteoblast.

Recent studies have revealed that the sympathetic nervous system is involved in bone metabolism. We previously reported that noradrenaline (NA) suppressed K(+) currents via Gi/o protein-coupled alpha1B-adrenergic receptor (α1B-AR) in human osteoblast SaM-1 cells. Additionally, it has been demonstrated that the intracellular Ca(2+) level ([Ca(2+)]i) was increased by NA via α1B-AR. In this study, we investigated the signal pathway of NA-induced [Ca(2+)]i elevation by using Ca(2+) fluorescence imaging in SaM-1 cells. NA-induced [Ca(2+)]i elevation was suppressed by pretreatment with a PLC inhibitor, U73122. This suggested that the [Ca(2+)]i elevation was mediated by Gq protein-coupled α1B-AR. On the other hand, NA-induced [Ca(2+)]i elevation was completely abolished in Ca(2+)-free solution, which suggested that Ca(2+) influx is the predominant pathway of NA-induced [Ca(2+)]i elevation. Although the inhibition of K(+) channel by NA caused membrane depolarization, the [Ca(2+)]i elevation was not affected by voltage-dependent Ca(2+) channel blockers, nifedipine and mibefradil. Meanwhile, NA-induced [Ca(2+)]i elevation was abolished following activation of store-operated Ca(2+) channel by thapsigargin. Additionally, the [Ca(2+)]i elevation was suppressed by store-operated channel inhibitors, 2-APB, flufenamate, GdCl3 and LaCl3. These results suggest that Ca(2+) influx through store-operated Ca(2+) channels plays a critical role in the signal transduction pathway of Gq protein-coupled α1B-AR in human osteoblasts.

Acidosis inhibits mineralization in human osteoblasts.

Osteoblasts and osteoclasts maintain bone volume. Acidosis affects the function of these cells including mineral metabolism. We examined the effect of acidosis on the expression of transcription factors and mineralization in human osteoblasts in vitro. Human osteoblasts (SaM-1 cells) derived from the ulnar periosteum were cultured with α-MEM containing 50 µg/ml ascorbic acid and 5 mM ß-glycerophosphate (calcifying medium). Acidosis was induced by incubating the SaM-1 cells in 10 % CO2 (pH approximately 7.0). Mineralization, which was augmented by the calcifying medium, was completely inhibited by acidosis. Acidosis depressed c-Jun mRNA and increased osteoprotegerin (OPG) production in a time-dependent manner. Depressing c-Jun mRNA expression using siRNA increased OPG production and inhibited mineralization. In addition, depressing OPG mRNA expression with siRNA enhanced mineralization in a dose-dependent manner. Acidosis or the OPG protein strongly inhibited mineralization in osteoblasts from neonatal mice. The present study was the first to demonstrate that acidosis inhibited mineralization, depressed c-Jun mRNA expression, and induced OPG production in human osteoblasts. These results suggest that OPG is involved in mineralization via c-Jun in human osteoblasts.

Involvement of TRP channels in the signal transduction of bradykinin in human osteoblasts.

Bradykinin (BK), a mediator of pain and inflammation, is involved in bone metabolism. We have previously reported that BK increased the synthesis of interleukin-6 and prostaglandin E(2) via phosphorylation of ERK1/2 in human osteoblasts, SaM-1. In the present study, we investigated the signal transduction pathway of BK focusing on intracellular Ca(2+) kinetics in SaM-1 cells. Bath-applied BK increased intracellular Ca(2+) concentration through the activation of B(2) receptors. Removal of extracellular Ca(2+) attenuated the effects of BK. Additionally, thapsigargin, endoplasmic reticulum Ca(2+) pump inhibitor, completely inhibited BK-induced increase of intracellular Ca(2+). These results suggested that bath-applied BK activated store-operated Ca(2+) channels (SOCCs) following Ca(2+) store depletion via B(2) receptor. Although the molecular components of SOCCs have yet to be conclusively identified in all cell types, recent studies demonstrated that transient receptor potential canonical (TRPC) channels are candidates for them. TRPC1, TRPC3, TRPC4 and TRPC6 were expressed in SaM-1 cells and inhibitors of TRP channel, 2-aminoethoxydiphenyl borate, GdCl(3), LaCl(3) and flufenamic acid, inhibited the effects of BK. These findings suggested that BK activated SOCCs and induced Ca(2+) influx via B(2) receptor in human osteoblasts. Molecular components of the SOCCs are suggested to be TRPC channels.

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.

Inhibitory effect of chlorpromazine on RANKL-induced osteoclastogenesis in mouse bone marrow cells.

Chlorpromazine (CPZ), the first widely used phenothiazine tranquilizer, is shown to inhibit the action of intracellular calmodulin (CaM) and bone resorption in vivo and in vitro. In this study, CPZ (0.63 - 10 µM) dose-dependently inhibited the formation of tartrate-resistant acid phosphatase (TRAP) staining-positive osteoclast-like cells in mouse bone marrow cells (BMCs) treated with 1α,25(OH)(2)D(3) (10 nM) or soluble receptor activator of nuclear factor-κB ligand (s-RANKL) (20 ng/ml). Expressions of mRNA for the nuclear factor of activated T-cells c1 (NFATc1), a key regulator of osteoclast differentiation; dendritic cell-specific transmembrane protein (DC-STAMP), an essential protein for cell-cell fusion; and characteristic markers of osteoclasts such as TRAP, cathepsin K, carbonic anhydrase II, and calcitonin receptor in BMCs were up-regulated by s-RANKL and decreased by the addition of CPZ (5 µM) or the selective CaM antagonist W7, but not the inactive analog W5. The general CaM kinase (CaMK) inhibitor KN-93 and CaM-dependent phosphatase calcineurin inhibitor FK-506 also inhibited s-RANKL-induced osteoclastogenesis. Phenothiazines such as CPZ, trifluoperazine (TFPZ), and promethazine (PMZ) inhibited s-RANKL-induced osteoclast-like cell formation in mouse BMCs. Osteoclastogenesis inhibitory effects decreased in the order of TFPZ, CPZ, PMZ, depending on their anti-CaM potency. These findings suggest that CPZ inhibits RANKL-induced osteoclastogenesis by its anti-CaM action.

Hydrocortisone inhibits cellular proliferation by downregulating hepatocyte growth factor synthesis in human osteoblasts.

Glucocorticoids have multiple systemic effects that may influence bone metabolism but also directly affect osteoblasts by decreasing their proliferation. Using human osteoblastic SaM-1 cells, we examined whether the effects of hydrocortisone on cellular proliferation are mediated by hepatocyte growth factor (HGF). Human osteoblasts constitutively express both HGF and c-Met, its receptor. Hydrocortisone decreased the gene and protein expression of HGF as well as proliferation in SaM-1 cells. These hydrocortisone (0.01-1 µM)-induced decreases in HGF synthesis and cellular proliferation occurred in a concentration-dependent manner. However, no hydrocortisone (0.01-1 µM)-induced decrease in cellular proliferation was observed in human osteosarcoma-derived cells (HOS and SaOS-2), which are not able to produce HGF. In the cellular proliferation in SaM-1 cells, the decrease was blocked concentration-dependently by exogenously applied HGF (0.01-3 ng/ml). Furthermore, SU11274 (1 µM), a highly specific inhibitor of c-Met, suppressed the proliferation of SaM-1 cells, but not HOS cells. From these results, we concluded that hydrocortisone inhibits the proliferation of SaM-1 cells by interrupting the autocrine/paracrine loop via the downregulation of HGF synthesis.

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.

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.

Effects of propranolol on bone metabolism in spontaneously hypertensive rats.

The effects of propranolol (PRO), a nonselective beta-adrenergic receptor (beta-AR) antagonist with membrane-stabilizing action on bone metabolism, were examined in spontaneously hypertensive rats (SHR) showing osteoporosis with hyperactivity of the sympathetic nervous system. Treatment of SHR with PRO at 1 and 5 mg/kg p.o. for 12 weeks increased bone mass of the lumbar vertebra and proximal tibia without affecting blood pressure, but PRO at 50 and 100 mg/kg with hypotensive action did not increase bone mass. Next, the effects of PRO at 0.1, 1, and 10 mg/kg on bone status were examined in more detail. Compared with the SHR control, not only bone mass but also biomechanical parameters of strength and toughness of the lumbar vertebrae were increased in SHR treated with PRO at 0.1 and 1 mg/kg, suggesting antiosteoporotic action. PRO at 1 mg/kg statistically increased histomorphometry indices of bone formation, whereas PRO at doses of 0.1, 1, and 10 mg/kg decreased those of bone resorption. Antiosteoporotic effect of PRO is attenuated at 10 mg/kg compared with 0.1 and 1 mg/kg. In addition, treatment with timolol, a nonselective beta-AR antagonist without membrane-stabilizing action, or butoxamine, a selective beta2-AR antagonist, at 1 mg/kg increased bone mass in SHR. These results suggested that treatment of SHR with beta-blockers at low dose improved bone loss and bone fragility. This antiosteoporotic effect of beta-blockers seems to be caused by the blocking action of beta2-AR, regardless of the membrane-stabilizing action.