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.

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.

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.

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.

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.

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.

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.

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.

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.

Inhibitory effect of minocycline on osteoclastogenesis in mouse bone marrow cells.

OBJECTIVE: To study the effects of minocycline hydrochloride (MINO) on the formation of tartrate-resistant acid phosphatase (TRAP) staining-positive multinucleated osteoclast-like cells in mouse bone marrow cells (BMCs) treated with 1α,25(OH)(2)D(3) or soluble receptor activator of nuclear factor-κB ligand (s-RANKL). MATERIALS AND METHODS: Mouse BMCs were cultured in alpha-modified minimum essential medium containing foetal calf serum (10%) and tetracyclines (2.5, 5 and 10µM), such as MINO, tetracycline hydrochloride (TC), oxytetracycline hydrochloride (OXT) or doxycycline (DOXY) in the presence of 1α,25(OH)(2)D(3) (10nM) or s-RANKL (20ng/ml) for 7 days, and the number of TRAP staining-positive osteoclast-like cells was counted. In RNA isolated from BMCs treated with 1α,25(OH)(2)D(3) or s-RANKL in the presence or absence of MINO, the expressions of osteoclast differentiation relating to mRNA were analysed by reverse transcription-polymerase chain reaction. Cell viability was examined in mouse BMCs and rabbit osteoclasts treated with MINO (0.25-20µM and 2-50µM, respectively) for 24h. RESULTS: MINO, TC, OXT or DOXY inhibited 1α,25(OH)(2)D(3)-induced osteoclast-like cell formation in mouse BMCs dose dependently. MINO suppressed 1α,25(OH)(2)D(3)-induced up-regulation of mRNA expressions of TRAP, cathepsin K, carbonic anhydrase II, and calcitonin receptor, but not RANKL. MINO inhibited s-RANKL-induced osteoclast-like cell formation and up-regulation of mRNA expressions for nuclear factor of activated T-cells c1 (NFATc1), a key regulator of osteoclast differentiation; however, MINO had no effects on the viability of mouse BMCs and rabbit osteoclasts. CONCLUSION: MINO inhibits RANKL-induced osteoclastogenesis via down-regulation of NFATc1 mRNA expression in osteoclast precursor cells.

Inhibitory effect of vitamin K(2) on interleukin-1beta-stimulated proliferation of human osteoblasts.

The effect of the proinflammatory cytokine interleukin (IL)-1beta on the cellular proliferation of human osteoblastic cells (SaM-1) and osteosarcoma-derived cells (SaOS-2, HOS, and MG-63) was examined. IL-1beta stimulated the proliferation of SaM-1 and MG-63 cells, but had no effect on that of SaOS-2 or HOS cells. Using reverse transcription-polymerase chain reaction (RT-PCR) analysis, the mRNA expression of IL-1 receptor type I (IL-1R1) was detected in SaM-1 and MG-63 cells consistently, but not in SaOS-2 or HOS cells in the proliferative stage. Neither the decoy inhibitory IL-1 receptor type II (IL-1R2) nor IL-1R antagonist mRNA was detected in any of the cell lines, suggesting that IL-1beta stimulated proliferation via IL-1R1. The IL-1beta -stimulated proliferation was inhibited by the MAPK kinase (MEK) inhibitor PD98059 but not by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 or the cyclooxygenase-2 specific inhibitor NS-398, suggesting that IL-1beta stimulated proliferation via MEK, without affecting prostaglandin E(2) synthesis. IL-1beta stimulated cellular proliferation but inhibited the synthesis of osteocalcin containing gamma-carboxylated glutamic acid (Gla-OSCAL). Both the increased proliferation and decreased Gla-OSCAL synthesis were suppressed by vitamin K(2) (VK(2)), which is a cofactor for gamma-carboxylase. Furthermore, the inhibitory effect of VK(2) on IL-1beta -stimulated proliferation was suppressed by warfarin. However, rifampicin the nuclear receptor steroid and xenobiotic receptor (SXR) ligand had no effect of IL-beta, suggesting that IL-1beta is involved in VK(2) dependent gamma-calboxylation but not SXR-activation. These results suggest that IL-1beta stimulated cellular proliferation via MEK and inhibited Gla-OSCAL synthesis, which were both inhibited by VK(2) via gamma-carboxylation.

Demonstration of direct neurite-osteoclastic cell communication in vitro via the adrenergic receptor.

There is currently great interest in the bone metabolism induced by the sympathetic nerve system. Recently, direct neurite-osteoblastic cell communication was demonstrated using an in vitro co-culture model comprising neurite-sprouting murine superior cervical ganglia and MC3T3-E1 osteoblast-like cells. In the present study, we examined whether the direct nerve-osteoclastic cell communication was present in an in vitro co-culture model comprising cultured murine superior cervical ganglia and mouse osteoclast-like cells. RAW264.7 cells treated with receptor activator of NF-kappaB ligand were used as osteoclast-like cells. We found that the addition of scorpion venom (SV) elicited neurite activation via intracellular Ca(2+) mobilization and, after a lag period, osteoclastic Ca(2+) mobilization in the co-culture. SV did not have any direct effect on the osteoclastic cells in the absence of the neurites. The addition of an alpha(1)-adrenergic receptor (AR) antagonist, prazosin, concentration-dependently prevented the osteoclastic activation that resulted as a consequence of neural activation by SV. We also found that alpha(1)-adrenergic receptor agonists evoked transient Ca(2+) mobilization and gene expression of interleukin-6 in osteoclastic cells. These results demonstrate that osteoclastic activation occurs via alpha(1)-AR in osteoclastic cells as a direct response to neuronal activation.

Effect of bone morphogenetic protein-2 (BMP-2) or troglitazone, as an inducer of osteogenic cells or adipocytes, on differentiation of a bone marrow mesenchymal progenitor cell line established from temperature-sensitive (ts) simian virus (SV) 40 T-antigen gene transgenic mice.

TBR31-2 is one of the bone marrow stromal cell lines. Differentiation toward osteogenic cells and calcification was observed when TBR31-2 cells were cultured for 4 weeks. Bone morphogenetic protein-2 (BMP-2) stimulated alkaline phosphatase (ALP) activity in a dose- and time-dependent manner. On the other hand, troglitazone increased oil droplet accumulation in a dose-dependent manner. In the presence of BMP-2, an increase of expression in osteogenic cell differentiation marker genes and a decrease of expression in adipocyte differentiation marker genes were observed with the exception of the induced expression of peroxisome proliferator-activated receptor gamma (PPARgamma), however, troglitazone, a ligand of PPARgamma treatment exhibited the opposite tendency. Interestingly, treatment with both BMP-2 and troglitazone resulted in a decrease of ALP activity and an increase of oil droplet accumulation. Reverse tanscription-polymerase chain reaction (RT-PCR) analysis also indicated that osteogenic differentiation markers decreased and that adipocyte differentiation markers increased. Thus, when the cells were cultured with BMP-2, osteogenic differentiation was enhanced while the expression of PPARgamma was maintained, and the addition of troglitazone caused a significant number of differentiated cells into adipocytes. These findings indicate that BMP-2 enhanced osteogenic differentiation and the expression of adipogenic transcription factor (PPARgamma) followed by osteogenic differentiation without activation of PPARgamma by its ligand.

Electrophysiological properties of a novel Ca(2+)-activated K(+) channel expressed in human osteoblasts.

Intracellular Ca(2+) mobilization plays important roles in cell survival, proliferation, and differentiation of osteoblasts. In this study, we identified a novel type of Ca(2+)-activated K(+) channel in human osteoblasts and investigated its physiological roles. Using RT-PCR methods and single-channel analysis in the patch-clamp technique, we found that BK and IK channels were genetically expressed in human osteoblasts and had electrophysiological properties similar to those reported previously for the channels in other organs (conductance, voltage dependence, and sensitivity to intracellular Ca(2+)). Taking advantage of the fact that ATP induces elevation of the intracellular Ca(2+) concentration in human osteoblasts, we successfully demonstrated that ATP-induced hyperpolarization was effectively inhibited by the IK channel blockers charybdotoxin and clotrimazole and by a P2 purinergic receptor antagonist, suramin, but not by the BK channel blockers tetraethylammonium chloride and iberiotoxin under the current-clamp mode of whole-cell clamp. The present study is the first to demonstrate the electrophysiological properties and functional expression of IK channels in human osteoblasts, findings which suggest that IK channels are regulators of membrane potential that give rise to intracellular Ca(2+) mobilization by physiological stimulation.

Lysophosphatidic acid-stimulated interleukin-6 and -8 synthesis through LPA1 receptors on human osteoblasts.

Using human osteoblastic SaM-1 cells, we investigated the effects of lysophosphatidic acid (LPA) on the production of interleukin (IL)-6 and IL-8, molecules which are capable of stimulating the development of osteoclasts from their haematopoietic precursors, and examined the signal transduction systems involved in their effect on these cells. These human osteoblasts constitutively expressed endothelial differentiation genes (Edg)-2 and Edg-4, which are LPA receptors. LPA increased gene and protein expression of IL-6 and IL-8 in SaM-1 cells. The expression of IL-6 and IL-8 mRNAs was maximal at 1-3h, and the increase in IL-6 and IL-8 synthesis in response to lysophosphatidic acid (1-10 microM) occurred in a concentration-dependent manner. These increases were blocked by Ki16425, an Edg-2/7 antagonist. In addition, LPA caused an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)), which was inhibited by pretreatment with Ki16425 or 2-aminoethoxy-diphenylborate (2-APB), an inositol 1,4,5-triphosphate (IP(3)) receptor (IP(3)R) blocker. The pretreatment of SaM-1 cells with U-73122, a phospholipase C (PLC) inhibitor, and 2-APB also inhibited the increase in IL-6 and IL-8 synthesis in response to LPA. These findings suggest that extracellular LPA-induced IL-6 and IL-8 synthesis occurred through Edg-2 (LPA(1) receptor) and the activation of PLC and IP(3)-mediated intracellular calcium release in SaM-1 cells.

Inhibitory effect of NPY on isoprenaline-induced osteoclastogenesis in mouse bone marrow cells.

The effect of neuropeptide Y (NPY), a co-transmitter with noradrenaline in peripheral sympathetic nerve fibers, on the osteoclastogenesis in mouse bone marrow cell cultures treated with isoprenaline, a beta-adrenergic receptor (beta-AR) agonist, was examined. The mouse bone marrow cells constitutively expressed mRNAs for the NPY-Y1 receptor and beta2-AR. NPY inhibited the formation of osteoclast-like cells induced by isoprenaline but not that by 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) or soluble receptor activator of nuclear factor-kappaB ligand (RANKL); and it suppressed the production of RANKL and cyclic AMP (cAMP) increased by isoprenaline but not those increased by 1alpha,25(OH)2D3. NPY also inhibited osteoclastogenesis induced by forskolin, an activator of adenylate cyclase; however, it did not inhibit that induced by exogenously supplied dibutyryl cAMP, a cell-permeable cAMP analog that activates cAMP-dependent protein kinase. These results demonstrate that NPY inhibited the isoprenaline-induced osteoclastogenesis by blocking the agonist-elicited increases in the production of cAMP and RANKL in mouse bone marrow cells, suggesting an interaction between NPY and beta-AR agonist in bone resorption.

Cytokine-induced nitric-oxide-dependent apoptosis in mouse osteoblastic cells: involvement of p38MAP kinase.

The apoptotic signalling induced by pro-inflammatory cytokines was examined in mouse osteoblastic MC3T3-E1 cells. Annexin-V/propidium iodine double-staining analysis demonstrated that the combination of tumour necrosis factor-alpha, interleukin-1beta and interferon-gamma caused cell death in osteoblastic cells mediated by apoptosis, not necrosis. Treatment with these cytokines resulted in potent enhancement of inducible nitric-oxide synthase (iNOS) mRNA and nitric-oxide (NO) in the cells. A specific inhibitor of p38 mitogen-activated protein (MAP) kinase, i.e. SB203580, dose dependently inhibited the induction of iNOS mRNA, its enzyme product, NO and DNA fragmentation (as an apoptosis index) in the cytokine-treated cells (P<0.05). In contrast, PD98059, a specific inhibitor of MEK that acts immediately upstream of classic MAP kinase, had no effect on the induction of iNOS, NO or DNA fragmentation in the cells. These results demonstrate that this cytokine-induced apoptosis in mouse osteoblastic cells was mediated by a p38MAP-kinase-dependent iNOS system.