Ayami Kondo

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 β2-adrenergic activity towards both osteoblastic and osteoclastic cells. Decreased bone formation is based on the inhibition of osteoblastic activity through β2-adrenergic receptors on osteoblasts. Such findings indicate that β-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 β-blockers has been demonstrated to be associated with a reduced risk of fractures. These clinical studies suggest that pharmacological blockade of the β-adrenergic system is beneficial to the human skeleton. In another prospective study, however, no association between β-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 β-blockers and the pathogenesis of osteoporosis must be explored in detail.

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.

Involvement of nitric oxide and biopterin in proinflammatory cytokine-induced apoptotic cell death in mouse osteoblastic cell line MC3T3-E1.

We previously demonstrated that the addition of proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma) caused induction of mRNAs for inducible nitric oxide (NO) synthase and GTP cyclohydrolase I, a rate-limiting enzyme for 5,6,7,8-tetrahydrobiopterin (BH4) biosynthesis, and produced their end-products, NO and BH4, in osteoblastic cells. In the present study, we examined whether NO and BH4, biologically active substances produced in response to proinflammatory cytokines, are involved in the effect of these cytokines on cell viability and apoptotic cell death involving DNA fragmentation. Cytokines as well as S-nitroso-N-acetyl-d,l-penicillamine, an NO generator, decreased cell viability, whereas sepiapterin, which was converted intracellularly to BH4, increased it. The examination of cytotoxicity measured in terms of lactate dehydrogenase release and apoptotic cell death assessed by flow cytometric analysis showed that cytokine-induced reduction of cell viability may be based upon cell death by apoptosis, but not lytic death as in necrosis. In the presence of sepiapterin, cytokine treatment resulted in a statistically pronounced reduction in the amount of DNA fragmentation. Furthermore, this fragmentation could be blocked by 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide, an NO scavenger. These results suggest that cytokine-induced apoptotic cell death is attributed to NO and is protected by BH4, and that osteoblastic cells in response to proinflammatory cytokines operate both a stimulatory process resulting in NO production and an inhibitory one resulting in BH4 production for apoptotic cell death. Cytokine-induced apoptotic cell death may be a consequence of the predominance of the stimulatory process over the inhibitory process.

Coexpression of GTP cyclohydrolase I and inducible nitric oxide synthase mRNAs in mouse osteoblastic cells activated by proinflammatory cytokines.

Proinflammatory cytokines, a combination of IL‐1β, TNF‐α, and IFN‐γ, caused mRNA expression of GTP cyclohydrolase I (GTP‐CH), the rate‐limiting enzyme in tetrahydrobiopterin (BH4) biosynthesis, and of inducible nitric oxide synthase (iNOS) in a well‐characterized osteoblastic clone MC3T3‐E1 cell line. We found the expression of the GTP‐CH gene in osteoblasts for the first time. The expression of GTP‐CH and iNOS mRNAs was found to be maximal at 3 and 9 h, respectively. The expression of both genes elicited increases in BH4 and NO levels. Pharmacological studies using 2,4‐diamino‐6‐hydroxypyrimidine, an inhibitor of GTP‐CH activity, showed that BH4 is involved in the activity of iNOS, but not in the induction of iNOS mRNA. The results using an inhibitor of nuclear factor (NF)‐κB and activating protein‐1 (AP‐1) activation suggested that coinduction of the two genes in response to cytokines occurred via activation of NF‐κB and AP‐1. In MC3T3‐E1 cells BH4 and sepiapterin, producing BH4, could protect against apoptosis, i.e. the degradation of nuclear DNA in the cells, induced by NO derived from S‐nitroso‐N‐acetyl‐d,l‐penicillamine. These results suggest that the induction of BH4 together with NO by proinflammatory cytokines could protect against NO‐induced apoptosis in MC3T3‐E1 cells.

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.

Matrix metalloproteinase-3 in odontoblastic cells derived from ips cells: unique proliferation response as odontoblastic cells derived from ES cells.

We previously reported that matrix metalloproteinase (MMP)-3 accelerates wound healing following dental pulp injury. In addition, we reported that a proinflammatory cytokine mixture (tumor necrosis factor-α, interleukin (IL)-1β and interferon-γ) induced MMP-3 activity in odontoblast-like cells derived from mouse embryonic stem (ES) cells, suggesting that MMP-3 plays a potential unique physiological role in wound healing and regeneration of dental pulp in odontoblast-like cells. In this study, we tested the hypothesis that upregulation of MMP-3 activity by IL-1β promotes proliferation and apoptosis of purified odontoblast-like cells derived from induced pluripotent stem (iPS) and ES cells. Each odontoblast-like cell was isolated and incubated with different concentrations of IL-1β. MMP-3 mRNA and protein expression were assessed using RT-PCR and western blotting, respectively. MMP-3 activity was measured using immunoprecipitation and a fluorescence substrate. Cell proliferation and apoptosis were determined using ELISA for BrdU and DNA fragmentation, respectively. siRNA was used to reduce MMP-3 transcripts in these cells. Treatment with IL-1β increased MMP-3 mRNA and protein levels, and MMP-3 activity in odontoblast-like cells. Cell proliferation was found to markedly increase with no changes in apoptosis. Endogenous tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were constitutively expressed during all experiments. The exocytosis inhibitor, Exo1, potently suppressed the appearance of MMP-3 in the conditioned medium. Treatment with siRNA against MMP-3 suppressed an IL-1β-induced increase in MMP-3 expression and activity, and also suppressed cell proliferation, but unexpectedly increased apoptosis in these cells (P<0.05). Exogenous MMP-3 was found to induce cell proliferation in odontoblast-like cells derived from iPS cells and ES cells. This siRNA-mediated increase in apoptosis could be reversed with exogenous MMP-3 stimulation (P<0.05). Taken together, IL-1β induced MMP-3-regulated cell proliferation and suppressed apoptosis in odontoblast-like cells derived from iPS and ES cells.

Down-regulation of mTOR leads to up-regulation of osteoprotegerin in bone marrow cells.

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor regulates bone mass by inhibiting osteoclastic bone resorption. mTOR, which is the mammalian target of rapamycin, is a kinase and central regulator of cell growth, proliferation, and survival. By using Rapamycin, we studied whether mTOR pathway is associated with OPG protein production in the mouse bone marrow-derived stromal cell line ST2. Rapamycin markedly increased the level of soluble OPG in ST2 cells. This antibiotic treatment resulted in the suppression of phosphorylation of mTOR. Rapamycin had no effects on the proliferation, differentiation, or apoptosis of the cells. Treatment with bone morphogenetic protein-4, which can induce OPG protein in ST2 cells, also resulted in a decrease in the density of the phospho-mTOR-band, suggesting that the suppression of the phospho-mTOR pathway is necessary for OPG production in ST2 cells. Thus, suitable suppression of mTOR phosphorylation is a necessary requirement for OPG production in bone marrow stromal cells.

IL-1β-induced, matrix metalloproteinase-3-regulated proliferation of embryonic stem cell-derived odontoblastic cells is mediated by the Wnt5 signaling pathway.

We previously established a method for differentiating induced pluripotent stem cells and embryonic stem (ES) cells into α2 integrin-positive odontoblast-like cells. We also reported that interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation and suppresses apoptosis in these cells, suggesting that MMP-3 plays a potentially unique physiological role in the regeneration of odontoblast-like cells. Here, we examined whether up-regulation of MMP-3 activity by IL-1β was mediated by Wnt signaling and led to increased proliferation of odontoblast-like cells. IL-1β increased mRNA and protein levels of Wnt5a, Wnt5b and the Wnt receptor Lrp5. Exogenous Wnt5a and Wnt5b were found to increase MMP-3 mRNA, protein and activity, and interestingly the rate of proliferation in these cells. Treatment with siRNAs against Wnt5a, Wnt5b and Lrp5 suppressed the IL-1β-induced increase in MMP-3 expression and suppressed cell proliferation, an effect rescued by application of exogenous Wnt5. These results demonstrate the sequential involvement of Wnt5, Lrp5 and MMP-3 in effecting IL-1β-induced proliferation of ES cell-derived odontoblast-like cells.

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.

IL-1β-induced matrix metalloproteinase-13 is activated by a disintegrin and metalloprotease-28-regulated proliferation of human osteoblast-like cells.

We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloprotease) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1β induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7(+)hSMSC)-derived osteoblast-like (α7(+)hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations, however, IL-1β failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1β-induced MMP-13 expression is linked to this IL-1β-mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1β-induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7(+)hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7(+)hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA-induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation and apoptosis in α7(+)hSMSC-OB cells are regulated by ADAM-28.