Selective ß2-adrenergic Antagonist Butoxamine Reduces Orthodontic Tooth Movement.

Recently, involvement of the sympathetic nervous system in bone metabolism has attracted attention. ß2-Adrenergic receptor (ß2-AR) is presented on osteoblastic and osteoclastic cells. We previously demonstrated that ß-AR blockers at low dose improve osteoporosis with hyperactivity of the sympathetic nervous system via ß2-AR blocking, while they may have a somewhat inhibitory effect on osteoblastic activity at high doses. In this study, the effects of butoxamine (BUT), a specific ß2-AR antagonist, on tooth movement were examined in spontaneously hypertensive rats (SHR) showing osteoporosis with hyperactivity of the sympathetic nervous system. We administered BUT (1 mg/kg) orally, and closed-coil springs were inserted into the upper-left first molar. After sacrifice, we calculated the amount of tooth movement and analyzed the trabecular microarchitecture and histomorphometry. The distance in the SHR control was greater than that in the Wistar-Kyoto rat group, but no significant difference was found in the SHR treated with BUT compared with the Wistar-Kyoto rat control. Analysis of bone volume per tissue volume, trabecular number, and osteoclast surface per bone surface in the alveolar bone showed clear bone loss by an increase of bone resorption in SHR. In addition, BUT treatment resulted in a recovery of alveolar bone loss. Furthermore, TH-immunoreactive nerves in the periodontal ligament were increased by tooth movement, and BUT administration decreased TH-immunoreactive nerves. These results suggest that BUT prevents alveolar bone loss and orthodontic tooth movement via ß2-AR blocking.

Noradrenaline stimulates cell proliferation by suppressing potassium channels via G(i/o) -protein-coupled α(1B) -adrenoceptors in human osteoblasts.

BACKGROUND AND PURPOSE: Recent studies demonstrated that the sympathetic nervous system regulates bone metabolism via ß(2) -adrenoceptors. Although α-adrenoceptors are also expressed in osteogenic cells, their functions in bone metabolism have been less studied. We previously demonstrated that noradrenaline suppressed potassium currents via α(1B) -adrenoceptors in the human osteoblast SaM-1 cell line. The aim of this study was to investigate the signal transduction pathway and the physiological role of noradrenaline in human osteoblasts in more detail. EXPERIMENTAL APPROACH: To investigate signal transduction through α(1B) -adrenoceptors, we used whole-cell patch clamp recording and Ca fluorescence imaging. Potassium channels regulate membrane potential and cell proliferation activity in non-excitable cells, so we evaluated cell proliferation activity by BrdU incorporation and WST assay. KEY RESULTS: In SaM-1 cells, bath-applied noradrenaline elevated intracellular Ca(2+) concentration and this effect was abolished by both chloroethylclonidine, an α(1B) -adrenoceptor antagonist, and U73122, a PLC inhibitor. However, the inhibitory effect of noradrenaline on whole-cell current was unaffected by U73122. In contrast, in cells pretreated with either Pertussis toxin, a G(i/o) -protein-coupled receptor inhibitor, or gallein, a Gßγ-protein inhibitor, the inhibitory effect of noradrenaline on whole-cell current was significantly suppressed. Noradrenaline-induced enhancement of cell proliferation was inhibited by CsCl, a non-selective potassium channel blocker, gallein and H89, a PKA inhibitor, but not by U73122. CONCLUSIONS AND IMPLICATIONS: Noradrenaline facilitated cell proliferation by regulation of potassium currents in human osteoblasts via G(i/o) -protein-coupled α(1B) -adrenoceptors, not via coupling to Gq-proteins.

Increase in RANKL: OPG ratio in synovia of patients with temporomandibular joint disorder.

Although a recent study suggested the involvement of RANKL and osteoprotegerin (OPG) in the pathogenesis of bone-destructive disease, no study has focused on the RANKL:OPG ratio in the synovial fluid of patients with temporomandibular joint (TMJ) disorder. This communication reports on the concentrations of RANKL and OPG in synovial fluid from TMJ patients and healthy control individuals. In contrast to an unchanged concentration of RANKL, a strong decrease in the concentration of OPG was detected in the synovial fluid from patients with TMJ internal derangement. Treatment with the synovial fluid of osteoarthritis (OA) patients resulted in the high production of osteoclast-like cells from blood mononuclear cells in vitro, as well as in pit formation in dentin slices. The addition of anti-RANKL IgG or OPG attenuated OA-synovial fluid-induced osteoclast formation, suggesting that the increase in the RANKL:OPG ratio in the microenvironment of the joint has the potential to induce osteoclastogenesis in TMJ osteoarthritis.

Dual roles for NF-kappaB activation in osteoblastic cells by serum deprivation: osteoblastic apoptosis and cell-cycle arrest.

To clarify the mechanisms of osteoblastic cell death, we examined whether serum deprivation would cause activation of the apoptotic signal cascade and arrest of the cell cycle in mouse osteoblastic MC3T3-E1 cells. Serum withdrawal from osteoblastic cell cultures resulted in growth arrest and cell-cycle arrest at G0/G1, which actions were accompanied by transient and potent activation of NF-kappaB, caspase-8, caspase-2, caspase-3, and caspase-9 in this order. Apoptosis, but not necrosis, in serum-deprived cells could be detected by FACS using Annexin-V/propidium iodine double staining. Serum deprivation also resulted in transient activation of the 20S proteasome, which is an important component for regulation of the cell cycle by the ubiquitin-proteasome system. The 20S proteasome inhibitor (PSI) but not NF-kappaB inhibitor SN50 suppressed the activation of proteasomes in serum-deprived cells. Although caspase inhibitors could not prevent the G0/G1 arrest in the serum-deprived cells, SN50 and the 20S proteasome inhibitor could block it. Since SN50, 20S proteasome inhibitor and caspase inhibitor could rescue cells from serum deprivation-induced apoptosis, the pathway for NF-kappaB/caspase activation is independent of the NF-kappaB/cell-cycle pathway, and the events downstream of the NF-kappaB/caspase-9 cascade lead to apoptosis. Taken together, our present results identify a novel role for NF-kappaB in cell-cycle and apoptosis regulation and underscore the significance of each independent signal cascade in serum-deprived osteoblastic cells.

Age-related changes of the ultrastructure in the cardiomyopathic hamster (UM-X7.1 Syrian hamster) parathyroid gland.

We qualitatively and quantitatively investigated parathyroid glands of the UM-X7.1 cardiomyopathic hamster at 1, 2, 6 and 12 months of age to compare them with those of the normal hamster. We found that at 1 month of age in the UM-X7.1 hamster, the Golgi apparatus, lipid droplets and secretory granules decreased. There were no significant differences between the UM-X7.1 hamster and the control hamster at 2 months of age. At 6 months of age, the Golgi apparatus, rER and the secretory granules significantly increased in the UM-X7.1 hamster. At 12 months of age, the Golgi apparatus and lysosomes increased, while the secretory granules decreased. Ultrastructurally, we consider that in the UM-X7.1 hamster, the synthesis and release of the parathyroid at 6 months of age may be activated by an excessive amount of circulating catecholamine, and the functional activity of the parathyroid glands at 12 months of age may be depressed by the increased plasma calcium level. These findings suggest that the activities of the synthesis and release of the parathyroid hormone were the highest at 6 months of age in the UM-X7.1 hamster.

Differential expression of RANKL and osteoprotegerin in gingival crevicular fluid of patients with periodontitis.

UNLABELLED: The receptor activator for NF-kappaB ligand (RANKL) plays an important role in osteoclast formation. A recent study with animal models suggests the involvement of RANKL in the pathogenesis of this periodontal disease. However, no one has examined the level of RANKL in the body fluid of human subjects. This communication reports on the in vivo concentrations of RANKL and the RANKL decoy receptor osteoprotegerin (OPG) in the gingival crevicular fluid (GCF) of periodontal subjects with severe, moderate, and mild forms of the disease. An increased concentration of RANKL and a decreased concentration of OPG were detected in GCF from patients with periodontitis (*p < 0.05 vs. control subjects). The ratio of the concentration of RANKL to that of OPG in the GCF was significantly higher for periodontal disease patients than for healthy subjects (*p < 0.01). Taken together, these data suggest that RANKL and OPG contribute to osteoclastic bone destruction in periodontal disease. ABBREVIATIONS: GCF, gingival crevicular fluid; IL, interleukin; OPG, osteoprotegerin; RANKL, receptor activator for NF-kappaB ligand.

Involvement of FGF-2 in the action of Emdogain on normal human osteoblastic activity.

OBJECTIVE: The present study was designed to evaluate the pharmacological characteristics of Emdogain (EMD) on cell growth and cell activity in human osteoblasts. METHODS: Cell proliferation as well as several gene and protein expressions were examined using reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) procedures in human osteoblastic cells (SaM-1) treated with EMD (30 microg ml(-1)). RESULTS: Treatment of osteoblasts with EMD significantly stimulated cell proliferation and fibroblast growth factor (FGF)-2 expression but decreased alkaline phosphatase expression. In addition, increases in cyclooxygenase (COX)-2 expression and decreases in matrix metalloproteinases (MMP)-1 expression were observed in osteoblasts treated with EMD. The effects of EMD on FGF-2 and MMP-1 expressions were not observed in osteoblasts treated with NS-398, an inhibitor of COX-2. The decrease in MMP-1 mRNA by EMD was prevented by treatment with antisense oligodeoxynucleotide (AS-ODN) for FGF-2. CONCLUSION: Emdogain showing both stimulation of cell proliferation and inhibition of cell differentiation has been shown to increase FGF-2 expression in the mediation of prostaglandin E2 and to decrease MMP-1 mRNA expression through the activation of FGF-2. FGF-2 may underlie in the action of EMD on osteoblasts during periodontal regeneration.

Fgf-2 regulates enamel and dentine formation in mouse tooth germ.

We examined the effects of basic fibroblast growth factor (FGF-2) on cultured lower molar tooth germ at the differentiative (bell) stage. Although FGF-2 has been detected in odontogenesis, its roles in biological activities, such as cell proliferation, differentiation and extracellular matrix mineralization are unclear. We assayed mRNA levels of the differentiation markers, dentine sialophosphoprotein (DSPP), amelogenin and alkaline phosphatase (ALP) using reverse transcription-polymerase chain reaction (RT-PCR), and histological methods. Tooth germs dissected from 17-day-old embryonic mice were cultured for 4 days with either recombinant human FGF-2 or specific antisense phosphorothioate oligodeoxynucleotide (antisense ODN) for FGF-2. Exogenous FGF-2 decreased the gene expression of differentiation markers in molars at the bell stage. Abrogation of endogenous FGF-2 by antisense ODN increased the gene expression of differentiation markers, and also significantly enhanced enamel and dentine formation. This histological change was recovered by adding exogeneous FGF-2. These findings suggest that FGF-2 at the bell stage regulates cell differentiation and matrix secretion.

Inhibitory effect of titanium particles on osteoclast formation generated by treatment of mouse bone marrow cells with PGE2.

OBJECTIVE: The present study was designed to evaluate the effect of titanium (Ti) particles with no endotoxin on osteoclast differentiation and osteoclast activity in in vitro experiments. METHODS: Osteoclast formation as well as osteoclastic bone resorbing activity were examined using the mouse bone marrow culture system and purified rabbit osteoclasts treated with Ti particles (2.5-20 microgram cm-2). RESULTS: Ti particles, with no adherent endotoxin, inhibited osteoclastogenesis and receptor activator of NF-kappaB ligand (RANKL) expression in bone marrow cells treated with prostaglandin E2 (PGE2) (100 nM). The inhibitory effect of Ti particles was concentration-dependent (5-20 microgram cm-2), and was observed only on the generation of osteoclasts by PGE2, but not by 1,25-dihydroxyvitamin D3 or soluble RANKL. This suggests that Ti particles did not act uniformly on a common process in the generation of osteoclasts, but specifically on signal transduction for PGE2 in generating osteoclasts. In highly purified osteoclasts, Ti particles showed no effect on survival and bone resorbing activity. CONCLUSION: Ti particles inhibited osteoclast differentiation and RANKL expression in mouse bone marrow cells treated with PGE2, without affecting mature osteoclast survival or activity. Thus, Ti particles may alter the osteoclastogenetic action of PGE2, which is one of the regulatory factors of bone remodeling.

Signal transduction system for interleukin-6 synthesis stimulated by lipopolysaccharide in human osteoblasts.

Lipopolysaccharide (LPS) is a bacterial cell component that plays multifunctional roles in inflammatory reactions, and one of the roles is as a powerful stimulator of bone resorption. LPS stimulated bone resorption via CD14 in mouse calvaria and was reported to function as a receptor for bacterial LPS complexed with serum proteins. Interleukin-6 (IL-6) is capable of stimulating the differentiation of osteoclasts from their hematopoietic precursors, and LPS elevates IL-6 synthesis in human osteoblastic cells. However, the signaling pathway of LPS-induced IL-6 synthesis in osteoblasts is unknown. In the present study, we could detect the existence of CD14 in human osteoblastic cells by RT-PCR analysis and show that LPS increased IL-6 mRNA and synthesis via CD14 in human osteoblastic cells. In human osteoblasts (SaM-1 cells) treated with 10 microg/ml LPS, increases in IL-6 mRNA and synthesis were inhibited by anti-CD14 antibody (MEM-18), PD98059 (an inhibitor of classic mitogen-activated protein kinase [MAPK]), or SB203580 (an inhibitor of p38 MAPK) but were not inhibited by H-89 (an inhibitor of protein kinase A [PKA]) and calphostin C (an inhibitor of protein kinase C [PKC]). Furthermore, LPS-induced IL-6 synthesis was inhibited by curcumin (an inhibitor of activating protein-1 [AP-1]) but not by pyrrolidine dithiocarbamate (PDTC) (an inhibitor of nuclear factor kappa B [NF-kappaB]). The findings of the present study suggest that the LPS receptor CD14, existent in human osteoblastic cells, and IL-6 synthesis in response to LPS probably occur via CD14, p38 MAPK, and MAP kinase/extracellular-regulated kinase kinase (MEK), leading to the transcriptional activation of AP-1 in human osteoblastic cells.

Histopathologic features of masseter muscle in the distrophic hamster (UM-X7.1 Syrian hamster).

Dystrophic hamster has been regarded as the useful model animal for Severe childhood autosomal recessive muscular dystrophy (SCARMD). Although, many studies on Dystrophic hamster have utilized the muscular tissue of the trunk, however no study have been analyzed for the masticatory muscle. For this study, we used a Dystrophic hamster (UM-X7.1 Syrian hamster) to histochemically investigate the effect of muscular dystrophy on the masseter muscle. Large and small regenerated muscle fibers, and necrotic fibers were detected almost in all areas. Opaque fiber, hypertrophic fiber with fiber splitting structure and necrotic fiber filled up by mononuclear phagocytes were recognized. The region, in which the mononuclear phagocytic cells infiltrated, showed strong positivity to acid phosphatase, and lysosome enzyme. There were many muscle fibers with reduced levels of succinate dehydrogenase (SDH) activities in the muscle fiber. Some TUNEL-positive cells were confirmed in both necrotic and non-necrotic areas. It was suggested that a part of TUNEL-positive cells are the cells originated from the connective tissue or immunocytes. In this result, histopathologic changes of the masseter muscle of the UM-X7.1 Syrian hamster was similar to muscle of the body trunk in the past reports. As the result, it was suggested that jaw closing movements may be negatively affected caused by the decline of the masseter muscle twitch. And, the point of view by which apoptosis is the trigger for the muscle fiber collapse were not seen in the Dystrophic hamster masseter muscle. We suggest that apoptosis is a one step in the process of regeneration of muscle fibers.

Activated human T cells directly induce osteoclastogenesis from human monocytes: possible role of T cells in bone destruction in rheumatoid arthritis patients.

OBJECTIVE: To elucidate the direct role of human T cells in the induction of osteoclastogenesis in rheumatoid arthritis (RA), by studying human monocytes and the pathogenetic roles of receptor activator of nuclear factor kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG). METHODS: Synovial tissue obtained at total knee replacement was stained immunohistologically using anti-RANKL, CD3, and CD4 antibodies. Synovial fluid was obtained from patients with RA, osteoarthritis (OA), gout, or trauma. Concentrations of the soluble form of RANKL (sRANKL) and OPG in the synovial fluid were measured by enzyme-linked immunosorbent assay. Activated T cells from peripheral blood mononuclear cells (PBMC) of healthy volunteers were cultured with human monocytes from PBMC. RESULTS: Immunostaining of the synovial tissue of RA patients demonstrated that RANKL-positive cells were detected in a subset of fibroblast-like synoviocytes and infiltrating mononuclear cells. Double immunostaining revealed that RANKL-positive cells were detected in a subset of CD3+ cells and CD4+ cells. An increased concentration of sRANKL and a decreased concentration of OPG were detected in synovial fluid from RA patients. The ratio of the concentration of sRANKL to that of OPG was significantly higher in synovial fluid of RA patients than in synovial fluid of patients with OA or gout. The activated T cells expressing RANKL induced osteoclastogenesis from autologous peripheral monocytes. The role of RANKL in this osteoclastogenetic process was confirmed by dose-dependent inhibition by OPG. CONCLUSION: The present study is the first to demonstrate osteoclastogenesis using human-derived T cells and monocytes. In addition, the present findings suggest that excess production of RANKL by activated T cells increases the level of sRANKL in synovial fluid and may contribute to osteoclastic bone resorption in RA patients.

Adrenergic stimulation of osteoclastogenesis mediated by expression of osteoclast differentiation factor in MC3T3-E1 osteoblast-like cells.

It is well known that adrenergic agonists efficiently activate beta-adrenoceptors on osteoblastic cells and can stimulate bone resorption in intact mouse calvaria. Recently, an osteoclastogenic factor of osteoblastic origin was found to be a novel tumor necrosis factor ligand family member and was termed osteoclast differentiation factor (ODF). Using a reverse transcription-polymerase chain reaction approach, we investigated the effect of epinephrine on mRNA levels of ODF and its decoy receptor, osteoclastogenesis inhibitory factor (OCIF), in MC3T3-E1 cells. Treatment with epinephrine (1 microM) rapidly increased ODF and OCIF mRNA levels, which peaked after 0.5 hr of treatment. Epinephrine (1 microM) also increased interleukin (IL)-6, IL-11, and cyclooxygenase (COX)-II mRNA levels, as well as increased prostaglandin E(2) (PGE(2)) accumulation in the culture medium. Treatment of the cells with IL-11 (10 ng/mL) or PGE(2) (1 microM) increased ODF and OCIF mRNA levels as observed with epinephrine. However, increases in ODF and OCIF mRNA levels by epinephrine were more rapid than those by IL-11, and were not influenced by NS-398 (100 microM; an inhibitor of COX-II), suggesting a direct effect of epinephrine on ODF and OCIF mRNA expressions as well as an indirect effect mediated by IL-11 and PGE(2) production. Epinephrine-induced increases in ODF and OCIF mRNA levels were inhibited by pretreatment with timolol (1 microM; beta-antagonist) and phentolamine (1 microM; alpha-antagonist), respectively. Furthermore, the formation of tartrate-resistant acid phosphatase-positive multinucleated cells from mouse bone marrow cells was stimulated by isoproterenol (0.1 to 10 microM) or epinephrine (0.1 to 10 microM). The action of isoproterenol, a beta-agonist, was clearly stronger than that of epinephrine, suggesting the importance of the physiological balance between ODF and OCIF productions for osteoclastogenesis. These findings suggest that beta-adrenergic stimulation induces not only IL-6, IL-11, and PGE(2) but also ODF expression in osteoblastic cells, leading to a stimulation of osteoclastogenesis.

Signal transduction system for interleukin-6 and interleukin-11 synthesis stimulated by epinephrine in human osteoblasts and human osteogenic sarcoma cells.

Epinephrine increased gene- and protein-expression of interleukin-6 (IL-6) and interleukin-11 (IL-11), which are capable of stimulating the development of osteoclasts from their hematopoietic precursors, in human osteoblast (SaM-1) and human osteosarcoma (SaOS-2, HOS, and MG-63) cell lines. An increase in IL-6 and IL-11 synthesis in response to epinephrine appeared to be a common feature in osteoblastic cells, but the magnitude of expression was different in these cell lines. In HOS cells treated with epinephrine, increases of IL-6 and IL-11 synthesis were inhibited by timolol (a beta-blocker), H-89 (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide; an inhibitor of protein kinase A (PKA)) and SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; an inhibitor of p38 mitogen-activated protein kinase (MAPK)], but not by phentolamine (an alpha-blocker), calphostin C [an inhibitor of protein kinase C (PKC)], or PD98059 (2'-amino-3'-methoxyflavone; an inhibitor of classic MAPK), suggesting a common pathway mediated by beta-adrenergic receptors in the PKA and p38 systems involved in the signal transduction of IL-6 and IL-11. Furthermore, expression of both genes was inhibited by curcumin [an inhibitor of activating protein-1 (AP-1) activation], but not by pyrrolidine dithiocarbamate (PDTC) [an inhibitor of nuclear factor (NF)-kappaB]. The pharmacological study suggested that coinduction of the two genes in response to epinephrine occurred via activation of AP-1. The findings of the present study suggest that coinduction of IL-6 and IL-11 in response to epinephrine probably occurs via the PKA and p38 MAPK systems, leading to the transcriptional activation of AP-1 in human osteoblastic cells.

Glial cell line-derived neurotrophic factor in the substantia nigra from control and parkinsonian brains.

Glial cell line-derived neurotrophic factor (GDNF) was measured for the first time in the brain (substantia nigra, caudate nucleus, putamen, cerebellum, and frontal cortex) from control and parkinsonian patients by highly sensitive sandwich enzyme-linked immunosorbent assay. In both groups, the levels of GDNF in the various brain regions were lower (pg/mg protein) than those of brain-derived growth factor (ng/mg order), and were significantly higher in the nigro-striatal dopaminergic regions (substantia nigra, caudate nucleus, putamen) than in the cerebellum and frontal cortex (P < 0.05). However, the content of GDNF in the dopaminergic regions showed no significant difference between parkinsonian and control patients.

Cytokines in Parkinson's disease.

We found that in Parkinson's disease (PD) the levels of various cytokines [tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-2, IL-4, IL-6, epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, TGF-beta1] were significantly increased in the striatum (caudate and putamen) of the postmortem brain and in ventricular or spinal cerebrospinal fluid (VCSF, LCSF). Furthermore, the levels of the apoptosis-related proteins such as bcl-2 and soluble Fas (sFas) in the striatum were also elevated in PD. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated parkinsonism mice, the levels of IL-1beta in the striatum were significantly increased, but those of nerve growth factor (NGF) were significantly decreased, compared with control mice. In hemiparkinsonism rats produced by injection of 6-hydroxydopamine (6-OHDA) into one side of the median forebrain bundle, the levels of TNF-alpha in the 6-OHDA-treated side were increased in the striatum and substantia nigra, but not in the cerebral cortex, compared with those in the control side. Repeated administration of L-DOPA in the 6-OHDA-treated rats did not change the TNF-alpha levels in the control side and in the 6-OHDA-treated side in the substantia nigra, striatum, and cerebral cortex. Our results suggest that the changes in the levels of cytokines, neurotrophins, and apoptosis-related proteins in the nigrostriatal regions of PD may be involved in apoptosis and degeneration of the nigrostriatal DA neurons.

Osteoclast differentiation factor in human osteosarcoma cell line.

A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for human osteoclast differentiation factor (ODF/RANKL/ OPGL/TRANCE) utilizing a polyclonal antibody that recognizes both human soluble ODF and mouse ODF in combination with a osteoclasogenesis inhibitory factor (OCIF/OPG) was developed. We can quantify the ODF level in not only human ODF (detection limit: 0.05 ng/ml), but also mouse ODF by virtue of cross-reactivity. Employing this assay system, we demonstrated that ODF is constitutively present as a membrane-bound form in both the human osteosarcoma cell lines, MG-63, HOS and SaOS-2, and the mouse osteoblastic cell line MC3T3-E1.

Expression of mRNA for axon guidance molecules, such as semaphorin-III, netrins and neurotrophins, in human osteoblasts and osteoclasts.

In the present study, we demonstrated the constitutive expression of diffusible axon guidance molecules such as neurotrophins, semaphorin-III, netrin-1, and netrin-2-like protein, which are known to function as a chemoattractant and/or chemorepellent for growing nerve fibers, in human osteoblastic and osteoclastic cells. The findings, obtained by RT-PCR, ELISA, and Western blot analysis suggest the extension of axons of peripheral sensory and sympathetic neurons to osteoblastic and osteoclastic cells and the possible neural regulation of bone metabolism in these osteogenic cells.

Increase in level of tumor necrosis factor-alpha in 6-hydroxydopamine-lesioned striatum in rats is suppressed by immunosuppressant FK506.

We compared in rats with 6-hydroxydopamine (6-OHDA)-induced hemiparkinsonism the content of tumor necrosis factor (TNF)-alpha in the nigrostriatal dopaminergic region of the control side with that of the 6-OHDA-injected experimental side, and explored the effects of 6-OHDA injection combined with the immunosuppressant FK506 treatment (0.5 or 4 mg/kg per day for 2 weeks). The ratios of the concentration of TNF-alpha in the striatum and substantia nigra on the 6-OHDA injection side to that on the control side in the 6-OHDA hemiparkinsonism rats were significantly higher than those in the control rats without 6-OHDA treatment, whereas those in the rats treated with 6-OHDA and FK506 were not significantly different from those in the control rats. Thus FK506 attenuated increased TNF-alpha level in the nigrostriatal dopaminergic region injured by 6-OHDA treatment.

Anti-apoptotic action of nerve growth factor in mouse osteoblastic cell line.

We investigated the potential role of nerve growth factor (NGF) in osteoblast survival in vitro. We found the expression of the mRNAs encoding NGF, brain-derived neurotrophic factor (BDNF), and trk-b, which is the receptor molecule of BDNF in mouse osteoblastic MC3T3-E1 cells. NGF high-affinity receptor trk-a was expressed continuously in the cells as visualized by Western blotting. A proinflammatory cytokine mixture stimulated NGF mRNA, and NGF protein release from MC3T3-E1 cells. When the effect of the nuclear factor-KB inhibitor pyrrolidine dithiocarbamate (PDTC) and activating protein-1 inhibitor curcumin were examined, a dose-dependent inhibition of cytokine-activated NGF expression occurred in the presence of PDTC or curcumin. Further, a specific inhibitor of p38 mitogen activated protein kinase (MAPK), i.e., SB203580, inhibited the induction of NGF in cytokines-treated cells in a dose-dependent manner whereas a specific inhibitor of classic MAPK, PD98059 had no effect on the induction of NGF. Treatment of anti-NGF IgG resulted in a potent increase of DNA fragmentation at a dose-dependent manner. NGF but not BDNF caused a dose-dependent reduction in the extent of apoptotic DNA breakdown under treatment with cytokines. Under similar conditions, the addition of NGF resulted in a potent reduction in bax protein but not in Fas, or bcl-xl. These findings demonstrated that NGF in non-neuronal osteoblastic cells may play an important role in cell survival as an anti-apoptotic factor.