Biomedical subjects

Akifumi Togari

Publications and source records attributed to Akifumi Togari.

15 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

[Increased bone resorption by sympathetic nervous activity].

Recent experimental studies showed the dynamic neural regulation of local bone metabolism. Increased sympathetic nervous activity has been demonstrated to cause bone loss via an increase in bone resorption and a decrease in bone formation. This review summarized both in vivo and in vitro evidences implicating sympathetic neuron action in bone resorption.

Animals↗

PubMed2005

Inhibitory effect of CGRP on osteoclast formation by mouse bone marrow cells treated with isoproterenol.

The present study was designed to elucidate the mode of action of isoproterenol (Isp; adrenergic beta-agonist) and to characterize the effect of the calcitonin gene-related peptide (CGRP; sensory neuropeptide) on osteoclast formation induced by Isp in a mouse bone marrow culture system. Treatment of mouse bone marrow cells with Isp generated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs) capable of excavating resorptive pits on dentine slices, and caused an increase in receptor activator of NF-kappaB ligand (RANKL) and a decrease in osteoprotegerin (OPG) production by the marrow cells. The osteoclast formation was significantly inhibited by OPG, suggesting the involvement of the RANKL-RANK system. CGRP inhibited the osteoclast formation caused by Isp or soluble RANKL (s-RANKL) but had no influence on RANKL or OPG production by the bone marrow cells treated with Isp, suggesting that CGRP inhibited the osteoclast formation by interfering with the action of RANKL produced by the Isp-treated bone marrow cells without affecting RANKL or OPG production. This in vitro data suggest the physiological interaction of sympathetic and sensory nerves in osteoclastogenesis in vivo.

Adrenergic beta-Agonists↗

PubMed2005

ATP-stimulated interleukin-6 synthesis through P2Y receptors on human osteoblasts.

We investigated the effect of extracellular adenosine triphosphate (ATP) on the production of interleukin (IL)-6, whose molecules are capable of stimulating the development of osteoclasts from their hematopoietic precursors as well as are involved in signal transduction systems in human osteoblastic SaM-1 cells. These human osteoblasts constitutively expressed P2X4, P2X5, P2X6, P2Y2, P2Y5, and P2Y6 purinergic receptors. ATP increased gene- and protein-expression of IL-6 in SaM-1 cells. The expression of the IL-6 mRNA was maximal at 1h, and the increase in IL-6 synthesis in response to ATP (10-100 microM) occurred in a concentration-dependent manner. Over the same concentration range of the nucleotide that was effective for IL-6 synthesis, ATP caused an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)), which increase was inhibited by pretreatment with suramin, a P2Y receptor antagonist, or 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate receptor blocker, but not by the extracellular Ca(2+)-chelating agent EGTA. The pretreatment of SaM-1 cells with suramin or 2-APB also inhibited the increase in IL-6 synthesis in response to ATP. These findings suggest that extracellular ATP-induced IL-6 synthesis occurs through P2Y receptors and mobilization of Ca(2+) from internal stores in human osteoblastic cells.

Adenosine Triphosphate↗

PubMed2005

IFN-gamma-producing human T cells directly induce osteoclastogenesis from human monocytes via the expression of RANKL.

The current study explored our hypothesis that IFN-gamma-producing human T cells inhibit human osteoclast formation. Activated T cells derived from human PBMC were divided into IFN-gamma-producing T cells (IFN-gamma(+) T cells) and IFN-gamma-non-producing T cells (IFN-gamma(-) T cells). IFN-gamma(+) T cells were cultured with human monocytes in the presence of macrophage-CSF alone. The concentration of soluble receptor activator of NF-kappaB ligand (RANKL) and IFN-gamma, and the amount of membrane type RANKL expressed on T cells, were measured by ELISA. In the patients with early rheumatoid arthritis (RA) treated with non-steroidal anti-inflammatory drugs alone, CD4+ T cells expressing both IFN-gamma and RANKL were detected by flow cytometry. Surprisingly, IFN-gamma(+) T cells, but not IFN-gamma(-) T cells, induced osteoclastogenesis from monocytes, which was completely inhibited by adding osteoprotegerin and increased by adding anti-IFN-gamma antibodies. The levels of both soluble and membrane type RANKL were elevated in IFN-gamma(+) T cells. The ratio of CD4+ T cells expressing both IFN-gamma and RANKL in total CD4+ T cells from PBMC was elevated in RA patients. Contrary to our hypothesis, IFN-gamma(+) human T cells induced osteoclastogenesis through the expression of RANKL, suggesting that Th1 cells play a direct role in bone resorption in Th1 dominant diseases such as RA.

Antirheumatic Agents↗

PubMed2005

The role of the sympathetic nervous system in controlling bone metabolism.

Experimental studies have generally shown that increased sympathetic nervous activity causes bone loss via an increase in bone resorption and a decrease in bone formation. Increased bone resorption is based on the stimulation of both osteoclast formation and osteoclast activity. These effects are associated with beta2-adrenergic activity towards both osteoblastic and osteoclastic cells. Decreased bone formation is based on the inhibition of osteoblastic activity through beta2-adrenergic receptors on osteoblasts. Such findings indicate that beta-blockers may be effective against osteoporosis, in which case there is increased sympathetic activity. In fact, in a population-based, case-control study, the current use of beta-blockers has been demonstrated to be associated with a reduced risk of fractures. These clinical studies suggest that pharmacological blockade of the beta-adrenergic system is beneficial to the human skeleton. In another prospective study, however, no association between beta-blocker use and fracture risk was shown in perimenopausal and older women. To confirm this important new therapeutic avenue to prevent bone loss, the relationship between the pharmacological effectiveness of beta-blockers and the pathogenesis of osteoporosis must be explored in detail.

Adrenergic Agonists↗

PubMed2004

Effect of tensile force on the expression of IGF-I and IGF-I receptor in the organ-cultured rat cranial suture.

Insulin-like growth factors (IGFs) play an important role in the regulation of bone metabolism. In this study, we investigated changes in the expression of IGF-I and IGF-I receptor and cell proliferation when a continuous tensile force was applied to the cranial suture of cultured rat calvaria. The parietal bones with the midsagittal suture were removed from male Wistar rats (19-days old), cultured for 24h, and divided into two groups. In the experimental group, tensile force (3 x 10(-3)N) was applied by helical springs to the midsagittal suture, whereas helical springs with no tension (0 N) were set in the control group. The tensile force significantly increased the expression of both IGF-I mRNA and protein (P < 0.05). By using in situ hybridisation, we also confirmed that IGF-I and IGF-I receptor mRNAs were localized in osteoblast-like and fibroblastic cells subjected to the tensile force. Also, this force stimulated the proliferation of osteoblast-like and fibroblastic cells in calvaria, without affecting their alkaline phosphatase activity. These results indicate that a tensile force applied to a cranial suture can cause an increase in the production of IGF-I and IGF-I receptors in osteoblast-like and fibroblastic cells, and this increase in IGF-I may cause the proliferation of the cells in an autocrine or paracrine manner.

Animals↗

PubMed2004

Activation of osteoblastic functions by a mediator of pain, bradykinin.

We investigated the effects of bradykinin (BK) on the production of interleukin (IL)-6 and prostaglandin PGE(2), whose molecules are capable of stimulating the development of osteoclasts from their hematopoietic precursors as well as the signal transduction systems involved, in human osteoblasts (SaM-1 cells). BK receptors B1 (B1R) and B2 (B2R) were expressed in SaM-1 and osteosarcoma (SaOS-2, HOS, and MG-63) cells. Treatment of SaM-1 cells with BK increased the synthesis of both IL-6 and PGE(2) and the increase in both was blocked by HOE140 (B2R antagonist), but not by Des-Arg(9)-[Leu(8)]-BK (B1R antagonist). U-73122, a phospholipase C (PLC) inhibitor, suppressed BK-induced IL-6 and PGE(2) synthesis in SaM-1 cells. In addition, BK caused an increase in the intracellular Ca(2+) concentration ([Ca(2+)]i), which was inhibited by pretreatment with HOE140 or 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) blocker. Furthermore, both SB203580 (an inhibitor of p38 mitogen-activated protein kinase [MAPK]) and PD98059 (an inhibitor of MEK, upstream of ERK) attenuated the BK-induced IL-6 and PGE(2) synthesis. BK treatment resulted in the phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK)1/2, and 2-APB could suppress BK-induced phosphorylation of ERK1/2. These findings suggest that BK increased both IL-6 and PGE(2) synthesis in osteoblastic cells via B2R and that PLC, IP(3)-induced [Ca(2+)]i, MEK, and MAPKs were involved in the signal transduction in these cells.

Bradykinin↗

PubMed2003

Activation of caspases is required for osteoblastic differentiation.

Previous studies have shown that mouse osteoblastic MC3T3-E1 cells undergo apoptosis when exposed to a mixture of proinflammatory cytokines. Bone morphogenetic protein (BMP)s are important regulators of osteoblast differentiation. Because regulation of osteoblastic differentiation is poorly understood, we sought to determine if BMP-4-induced differentiation of osteoblastic cells depends on the activity of the key apoptotic proteases, i.e. the caspases. BMP-4 induced the growth arrest and differentiation of osteoblastic cell line MC3T3-E1, as evidenced by the appearance of osteoblastic phenotypes such as alkaline phosphatase (ALP) activation and parathyroid hormone (PTH)-dependent production of cAMP. Surprisingly, BMP-4 induced transient and potent activation of caspase-8, caspase-2, and caspase-3, in this order. However, no apoptosis or necrosis in BMP-4-treated cells could be detected by FACS using annexin-V/propodium iodine double staining. Peptide inhibition of caspase activity led to a dramatic reduction in ALP activation and PTH-induced production of cAMP in BMP-4-treated cells. Although BMP-4 treatment resulted in cell-cycle G0/G1 arrest as detected by FACS cell-cycle analysis, caspase inhibitors (caspase-8, caspase-2, and caspase-3 inhibitors) could block the G0/G1 arrest in MC3T3-E1 cells. Taken together, these results confirm a unique and unanticipated role for the caspase-mediated signal cascade in the differentiation of osteoblasts.

Alkaline Phosphatase↗

PubMed2003

Effects of beta-adrenergic agonists on bone-resorbing activity in human osteoclast-like cells.

In the present study, we demonstrate for the first time that beta-adrenergic agonists stimulate bone-resorbing activity in human osteoclast-like multinucleated cells (MNCs). Osteoclast-like MNCs constitutively expressed mRNA for alpha1B-, alpha2B- and beta2-adrenergic receptor (AR) in addition to characteristic markers of mature osteoclast, such as calcitonin receptor (CT-R), tartrate-resistant acid phosphatase (TRAP), alphaV-chain of integrin (Int alphaV), carbonic anhydrase II (CA-II) and cathepsin K (Cathe K). Epinephrine (1 microM; alpha,beta-adrenergic agonist) up-regulated expression of Int alphaV, CA-II and Cathe K in the osteoclast-like MNCs. Osteoclastic resorbing activity was markedly increased by isoprenaline (1 microM; beta-adrenergic agonist), moderately by epinephrine, but poorly by phenylephrine (1 microM; alpha1-adrenergic agonist). The actin ring, which was suggested to be correlated with bone-resorbing activity, was clearly observed in osteoclast-like MNCs treated with isoprenaline and epinephrine, but faintly in those treated with phenylephrine. These findings suggest that beta-adrenergic agonists directly stimulate bone-resorbing activity in matured osteoclasts.

Adrenergic beta-Agonists↗

PubMed2003

Activation of the peripheral sympathetic nervous system increased the expression of cyclooxygenase-2 (COX-2) mRNA in mouse calvaria.

Pharmacological stimulation of adrenoceptor was demonstrated to increase the synthesis of prostaglandin (PG)-E(2), well known to modulate bone metabolism by regulating development and function of osteoclasts and osteoblasts, in cultured osteoblastic cells. Recently, intracerebroventricular (i.c.v.) injection of lipopolysaccharide (LPS), which caused the inflammatory stimuli in the brain, was demonstrated to increase the outflow of the peripheral sympathetic nervous system. In this study, to clarify the physiological role of sympathetic nerves to bone metabolism in vivo, we examined the effect of LPS (i.c.v.) on the expression of cyclooxygenase-2 (COX-2) mRNA in mouse calvaria, using reverse transcription-polymerase chain reaction analysis. The expression of COX-2 mRNA was increased by LPS (i.c.v.) in mouse calvaria. The treatment with the neurotoxin 6-hydroxydopamine or beta-blocker inhibited the central LPS-induced COX-2 mRNA in mouse calvaria. In addition, the treatment of calvaria with isoprenaline, beta-agonist, or noradrenaline increased PGE(2) synthesis in the organ culture system. These findings show that central LPS-induced COX-2 mRNA was mediated by the activation of postganglionic sympathetic nerve fibers and beta-adrenoceptor in mouse calvaria and suggest that in vivo activation of the sympathetic nervous system modulates bone metabolism.

Animals↗

PubMed2003

Involvement of p38MAP kinase in bone morphogenetic protein-4-induced osteoprotegerin in mouse bone-marrow-derived stromal cells.

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor is a recently identified cytokine that belongs to the tumour necrosis factor receptor superfamily and regulates bone mass by inhibiting osteoclastic bone resorption. This study found that bone morphogenetic protein (BMP)-4 markedly increased the level of soluble OPG in the mouse bone-marrow-derived stromal cell line, ST2. In contrast, BMP-4 showed no effect on OPG ligand production in ST2 cells under similar conditions. Using an in vitro immunocomplex kinase assay, BMP-4 was found to activate p38 mitogen-activated protein (MAP) kinase. Pre-treatment of ST2 cells with SB203580 (a specific inhibitor of p38MAP kinase) inhibited BMP-4-induced increase in OPG, although PD98059 (a specific inhibitor of classic MAP kinase cascade) showed no effect on OPG production. These results clearly suggest that activation of the p38MAP kinase pathway is necessary for BMP-4-induced OPG induction in ST2 cells.

Animals↗

PubMed2003

In vivo stimulation of sympathetic nervous system modulates osteoblastic activity in mouse calvaria.

Previously, we demonstrated that epinephrine induced the expression of interleukin (IL)-6 mRNA via beta-adrenoceptors in cultured human osteoblastic cells. IL-6 is well known to modulate bone metabolism by regulating the development and function of osteoclasts and osteoblasts. Recently, restraint stress and intracerebroventricular injection of lipopolysaccharide (LPS) have been reported to induce the expression of IL-6 mRNA in peripheral organs in mice in which expression is mediated by the activation of the sympathetic nervous system. To prove the physiological role of sympathetic nerves in bone metabolism in vivo, we examined by RT-PCR analysis the effects of restraint stress and intracerebroventricular injection of LPS on IL-6 mRNA expression in mouse calvaria. The expression of IL-6 mRNA in mouse calvaria was stimulated by either restraint stress (30 min) or intracerebroventricular injection of LPS (50 ng/mouse, 60 min). The treatment of mice with the neurotoxin 6-hydroxydopamine (6-OHDA, 100 mg x kg-1 x day-1 ip for 3 days) inhibited LPS (icv)-induced expression of IL-6 mRNA in their calvaria. The expression of IL-6 mRNA induced by the restraint stress was not influenced by 6-OHDA, which destroys noradrenergic nerve terminals. Furthermore, pretreatment with a beta-blocker, propranolol (15 or 25 mg/kg ip), inhibited both stress- and LPS-induced increases in the level of IL-6 mRNA, but pretreatment with an alpha-blocker, phentolamine (5 mg/kg sc), did not inhibit them in mouse calvaria. In addition, treatment of calvaria with isoprenaline or norepinephrine increased IL-6 synthesis in the organ culture system. These results indicate that in vivo adrenergic stimulation modulates the osteoblastic activity in mouse calvaria via noradrenergic nerve terminals.

Animals↗

PubMed2002

Adrenergic regulation of bone metabolism: possible involvement of sympathetic innervation of osteoblastic and osteoclastic cells.

It has been demonstrated that human osteoblastic as well as osteoclastic cells are equipped with adrenergic receptors and neuropeptide receptors and that they constitutively express diffusible axon guidance molecules that are known to function as a chemoattractant and/or chemorepellent for growing nerve fibers. These findings suggest that the extension of axons of sympathetic and peripheral sensory neurons to osteoblastic and osteoclastic cells is required for the dynamic neural regulation of local bone metabolism. Recently, bone resorption modulated by sympathetic stimulation was demonstrated to be associated with ODF (osteoclast differentiation factor) and OCIF (osteoclastogenesis inhibitory factor) produced by osteoblasts/stromal cells. This review summarizes the evidence implicating sympathetic neuron action in bone metabolism. The possible function of osteoclastogenesis, which could result in the initiation of sympathomimetic bone resorption, is also discussed.

Axons↗

PubMed2002

Differential expression of the Fas-Fas ligand system on cytokine-induced apoptotic cell death in mouse osteoblastic cells.

Apoptotic signalling, particularly in the Fas-Fas ligand (FasL) system, was studied in a mouse osteoblastic cell line, MC3T3-E1. A combination of the cytokines tumour necrosis factor-alpha, interleukin-1beta and interferon-gamma activated the Fas-FasL-dependent cell-death system. The cytokines caused significant enhancement of Fas mRNA and Fas protein, and led to apoptotic cell death. Western blot demonstrated that FasL protein was continuously present in MC3T3-E1 cells, although the cytokines had no effect on the induction of FasL. Exogenous FasL caused a decrease in cell viability and a large increase in apoptotic cell death in cells pre-treated with cytokines, indicating that the Fas-FasL system has the potential to cause apoptosis in osteoblastic cells. Treatment with anti-Fas IgG (antagonistic antibody) inhibited the DNA fragmentation induced by cytokines in a dose-dependent manner, suggesting that cytokine-induced Fas may cause apoptotic cell death in MC3T3-E1 cells. Taken together, these findings show that cytokine-induced apoptotic cell death was mediated by the autocrine or paracrine Fas-FasL system in mouse osteoblastic cells, and suggest that cytokine-induced apoptosis could have an important role in localised bone destruction associated with inflammatory bone diseases such as periodontal disease.

3T3 Cells↗