Natsuko Tanabe

Effects of nicotine and lipopolysaccharide on the expression of matrix metalloproteinases, plasminogen activators, and their inhibitors in human osteoblasts

OBJECTIVE Lipopolysaccharide (LPS) from periodontopathic bacteria can initiate alveolar bone loss through the induction of host-derived cytokines. Smoking increases the risk and severity of periodontitis. We examined the effects of nicotine and LPS on the expression of matrix metalloproteinases (MMPs), plasminogen activators (PAs), and their inhibitors, including tissue inhibitors of metalloproteinases (TIMPs) and PA inhibitor-1 (PAI-1), in osteoblasts. METHODS The cells were cultured with or without 10(-4) M nicotine and 100 ng/ml LPS for 12 days or with 100 microg/ml polymyxin B, 10(-4) M D-tubocurarine, 10 micromol/ml NS398, or 10(-6) M celecoxib in the presence of either nicotine or LPS for 12 days. The gene and protein expression levels for MMPs, PAs, TIMPs, and PAI-1 were examined using real-time PCR and ELISAs, respectively. PGE(2) production was determined using an ELISA. RESULTS The addition of nicotine and/or LPS to the culture medium increased the expression of MMP-1, -2, and -3 and tissue-type PA (tPA); decreased the expression of TIMP-1, -3, and -4; and did not affect expression of TIMP-2 or PAI-1. In the presence of d-tubocurarine or polymyxin B, neither nicotine nor LPS stimulated the expression of MMP-1. In the presence of NS398 or celecoxib, the stimulatory effects of nicotine and LPS on MMP-1 expression were unchanged, but they were unable to stimulate PGE(2) production. CONCLUSION These results suggest that nicotine and LPS stimulate the resorption process that occurs during turnover of osteoid by increasing the production of MMPs and tPA and by decreasing the production of TIMPs. Furthermore, they suggest that the stimulatory effect of nicotine and LPS on PGE(2) production is independent of their stimulatory effect on MMP-1 expression.

Lysophosphatidic Acid Signals through Multiple Receptors in Osteoclasts to Elevate Cytosolic Calcium Concentration, Evoke Retraction, and Promote Cell Survival

Lysophosphatidic acid (LPA) is a bioactive phospholipid whose functions are mediated by multiple G protein-coupled receptors. We have shown that osteoblasts produce LPA, raising the possibility that it mediates intercellular signaling among osteoblasts and osteoclasts. Here we investigated the expression, signaling and function of LPA receptors in osteoclasts. Focal application of LPA elicited transient increases in cytosolic calcium concentration ([Ca2+]i), with 50% of osteoclasts responding at ∼400 nm LPA. LPA-induced elevation of [Ca2+]i was blocked by pertussis toxin or the LPA1/3 receptor antagonist VPC-32183. LPA caused sustained retraction of osteoclast lamellipodia and disrupted peripheral actin belts. Retraction was insensitive to VPC-32183 or pertussis toxin, indicating involvement of a distinct signaling pathway. In this regard, inhibition of Rho-associated kinase stimulated respreading after LPA-induced retraction. Real-time reverse transcription-PCR revealed transcripts encoding LPA1 and to a lesser extent LPA2, LPA4, and LPA5 receptor subtypes. LPA induced nuclear translocation of NFATc1 and enhanced osteoclast survival, effects that were blocked by VPC-32183 or by a specific peptide inhibitor of NFAT activation. LPA slightly reduced the resorptive activity of osteoclasts in vitro. Thus, LPA binds to at least two receptor subtypes on osteoclasts: LPA1, which couples through Gi/o to elevate [Ca2+]i, activate NFATc1, and promote survival, and a second receptor that likely couples through G12/13 and Rho to evoke and maintain retraction through reorganization of the actin cytoskeleton. These findings reveal a signaling axis in bone through which LPA, produced by osteoblasts, acts on multiple receptor subtypes to induce pleiotropic effects on osteoclast activity and function.

Osteopontin Signals through Calcium and Nuclear Factor of Activated T Cells (NFAT) in Osteoclasts A NOVEL RGD-DEPENDENT PATHWAY PROMOTING CELL SURVIVAL

Background: The extracellular matrix protein osteopontin enhances bone resorption by osteoclasts. Results: Osteopontin induces oscillations in the concentration of cytosolic calcium in osteoclasts, leading to nuclear translocation of transcription factor NFATc1 and increased survival. Conclusion: Osteopontin enhances osteoclast survival through a calcium-NFAT-dependent pathway. Significance: This novel mechanism explains in part the stimulatory effects of osteopontin on bone resorption. Osteopontin (OPN), an integrin-binding extracellular matrix glycoprotein, enhances osteoclast activity; however, its mechanisms of action are elusive. The Ca2+-dependent transcription factor NFATc1 is essential for osteoclast differentiation. We assessed the effects of OPN on NFATc1, which translocates to nuclei upon activation. Osteoclasts from neonatal rabbits and rats were plated on coverslips, uncoated or coated with OPN or bovine albumin. OPN enhanced the proportion of osteoclasts exhibiting nuclear NFATc1. An RGD-containing, integrin-blocking peptide prevented the translocation of NFATc1 induced by OPN. Moreover, mutant OPN lacking RGD failed to induce translocation of NFATc1. Thus, activation of NFATc1 is dependent on integrin binding through RGD. Using fluorescence imaging, OPN was found to increase the proportion of osteoclasts exhibiting transient elevations in cytosolic Ca2+ (oscillations). OPN also enhanced osteoclast survival. The intracellular Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) suppressed Ca2+ oscillations and inhibited increases in NFATc1 translocation and survival induced by OPN. Furthermore, a specific, cell-permeable peptide inhibitor of NFAT activation blocked the effects of OPN on NFATc1 translocation and osteoclast survival. This is the first demonstration that OPN activates NFATc1 and enhances osteoclast survival through a Ca2+-NFAT-dependent pathway. Increased NFATc1 activity and enhanced osteoclast survival may account for the stimulatory effects of OPN on osteoclast function in vivo.

Sodium butyrate stimulates mineralized nodule formation and osteoprotegerin expression by human osteoblasts

OBJECTIVE Butyric acid (sodium butyrate; BA) is a major metabolic by-product of main periodontopathic bacteria present in subgingival plaque. In the present study, we examined the effects of BA on cell proliferation, alkaline phosphatase (ALPase) activity, mineralized nodule formation, extracellular matrix protein expression, macrophage colony-stimulating factor (M-CSF), and osteoprotegerin (OPG) in normal human osteoblasts. METHODS The cells were cultured with 0, 10(-8), 10(-6) or 10(-4)M BA for up to 12 days. Mineralized nodule formation was detected by alizarin red staining, and the calcium content in mineralized nodules was determined using a calcium assay kit. The gene and protein expression levels for type I collagen, bone sialoprotein (BSP), osteopontin (OPN), M-CSF, and OPG were examined using real-time PCR and ELISA, respectively. RESULTS Mineralized nodule formation and the calcium content of mineralized nodules were increased by BA in a dose-dependent manner. Cell proliferation and ALPase activity were not affected by the addition of BA. Following the addition of 10(-4)M BA, the expression levels of BSP, OPN, and OPG increased, whereas the expression levels of type I collagen and M-CSF were not markedly affected. CONCLUSION These results suggest that BA stimulates bone formation by increasing the production of BSP and OPN, whereas it suppresses osteoclast differentiation by increasing the production of OPG by human osteoblasts.

Effects of Isoform-selective Phosphatidylinositol 3-Kinase Inhibitors on Osteoclasts ACTIONS ON CYTOSKELETAL ORGANIZATION, SURVIVAL, AND RESORPTION

Background: Little is known about the function of specific phosphatidylinositol 3-kinase (PI3K) isoforms in osteoclasts. Results: Using a panel of isoform-selective inhibitors, we found that PI3Kδ regulates osteoclast morphology, actin cytoskeletal organization, and resorptive activity. Conclusion: The PI3Kδ isoform plays a critical role in regulating osteoclast resorptive activity. Significance: PI3Kδ is an attractive target for anti-resorptive therapeutics. Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. The following inhibitors were investigated (targets in parentheses): wortmannin and LY294002 (pan-p110), PIK75 (α), GDC0941 (α, δ), TGX221 (β), AS252424 (γ), and IC87114 (δ). In addition, we characterized a new potent and selective PI3Kδ inhibitor, GS-9820, and explored roles of PI3K isoforms in regulating osteoclast function. Osteoclasts were isolated from long bones of neonatal rats and rabbits. Wortmannin, LY294002, GDC0941, IC87114, and GS-9820 induced a dramatic retraction of osteoclasts within 15–20 min to 65–75% of the initial area. In contrast, there was no significant retraction in response to vehicle, PIK75, TGX221, or AS252424. Moreover, wortmannin and GS-9820, but not PIK75 or TGX221, disrupted actin belts. We examined effects of PI3K inhibitors on osteoclast survival. Whereas PIK75, TGX221, and GS-9820 had no significant effect on basal survival, all blocked RANKL-stimulated survival. When studied on resorbable substrates, osteoclastic resorption was suppressed by wortmannin and inhibitors of PI3Kβ and PI3Kδ, but not other isoforms. These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics.

Nicotine Affects Bone Resorption and Suppresses the Expression of Cathepsin K, MMP-9 and Vacuolar-Type H+-ATPase d2 and Actin Organization in Osteoclasts

Tobacco smoking is an important risk factor for the development of several cancers, osteoporosis, and inflammatory diseases such as periodontitis. Nicotine is one of the major components of tobacco. In previous study, we showed that nicotine inhibits mineralized nodule formation by osteoblasts, and the culture medium from osteoblasts containing nicotine and lipopolysaccharide increases osteoclast differentiation. However, the direct effect of nicotine on the differentiation and function of osteoclasts is poorly understood. Thus, we examined the direct effects of nicotine on the expression of nicotine receptors and bone resorption-related enzymes, mineral resorption, actin organization, and bone resorption using RAW264.7 cells and bone marrow cells as osteoclast precursors. Cells were cultured with 10−5, 10−4, or 10−3 M nicotine and/or 50 µM α-bungarotoxin (btx), an 7 nicotine receptor antagonist, in differentiation medium containing the soluble RANKL for up 7 days. 1–5, 7, 9, and 10 nicotine receptors were expressed on RAW264.7 cells. The expression of 7 nicotine receptor was increased by the addition of nicotine. Nicotine suppressed the number of tartrate-resistant acid phosphatase positive multinuclear osteoclasts with large nuclei(≥10 nuclei), and decreased the planar area of each cell. Nicotine decreased expression of cathepsin K, MMP-9, and V-ATPase d2. Btx inhibited nicotine effects. Nicotine increased CA II expression although decreased the expression of V-ATPase d2 and the distribution of F-actin. Nicotine suppressed the planar area of resorption pit by osteoclasts, but did not affect mineral resorption. These results suggest that nicotine increased the number of osteoclasts with small nuclei, but suppressed the number of osteoclasts with large nuclei. Moreover, nicotine reduced the planar area of resorption pit by suppressing the number of osteoclasts with large nuclei, V-ATPase d2, cathepsin K and MMP-9 expression and actin organization.

Tension force-induced ATP promotes osteogenesis through P2X7 receptor in osteoblasts.

Orthodontic tooth movement induces alveolar bone resorption and formation by mechanical stimuli. Force exerted on the traction side promotes bone formation. Adenosine triphosphate (ATP) is one of the key mediators that respond to bone cells by mechanical stimuli. However, the effect of tension force (TF)‐induced ATP on osteogenesis is inadequately understood. Accordingly, we investigated the effect of TF on ATP production and osteogenesis in MC3T3‐E1 cells. Cells were incubated in the presence or absence of P2X7 receptor antagonist A438079, and then stimulated with or without cyclic TF (6% or 18%) for a maximum of 24 h using Flexercell Strain Unit 3000. TF significantly increased extracellular ATP release compared to control. Six percent TF had maximum effect on ATP release compared to 18% TF and control. Six percent TF induced the expression of Runx2 and Osterix. Six percent TF also increased the expression of extracellular matrix proteins (ECMPs), ALP activity, and the calcium content in ECM. A438079 blocked the stimulatory effect of 6% TF on the expression of Runx2, Osterix and ECMPs, ALP activity, and calcium content in ECM. This study indicated that TF‐induced extracellular ATP is released in osteoblasts, suggesting that TF‐induced ATP promotes osteogenesis by autocrine action through P2X7 receptor in osteoblasts. J. Cell. Biochem. 116: 12–21, 2015.

Low-intensity pulsed ultrasound-induced ATP increases bone formation via the P2X7 receptor in osteoblast-like MC3T3-E1 cells.

Low‐intensity pulsed ultrasound (LIPUS) is used for bone healing in orthopedics and dentistry. It has been shown that LIPUS induces the secretion of extracellular adenosine triphosphate (ATP), a key mediator of osteoblast response to mechanical stimuli. However, the detailed mechanism of LIPUS‐induced osteogenesis has been elusive. In this study, we investigated the role of the P2X7 receptor in LIPUS‐induced osteogenesis. LIPUS induced the release of extracellular ATP, differentiation of osteoblasts and osteogenesis via the P2X7 receptor, without affecting the activity of alkaline phosphatase (ALPase). These results suggest that LIPUS‐induced extracellular ATP promotes bone formation via the osteoblast P2X7 receptor independently of ALPase.

IL-1 Stimulates the Expression of Prostaglandin Receptor EP4 in Human Chondrocytes by Increasing Production of Prostaglandin E2

Prostaglandin (PG) E2, which exerts its actions via the PG receptors EP1–4, is produced from arachidonic acid by cyclooxygenase (COX)-1 and COX-2. The aim of this study was to investigate the mechanisms by which interleukin (IL)-1β induces the expression of PG receptors in cultured human chondrocytes and to explore the role of PGE2 in this process. The cells were cultured with 0, 10, or 100 U/mL IL-1β with or without 1 μM celecoxib, a specific inhibitor of COX-2, for up to 28 days. Expression of the genes encoding COX-1, COX-2, and EP1–4 was quantified using real-time PCR, and expression of the corresponding proteins was examined using immunohistochemical staining. PGE2 production was determined using ELISA. IL-1β treatment caused a marked dose- and time-dependent increase in the levels of PGE2, COX-2, and EP4 as compared with the untreated control. It did not affect the expression of COX-1, and it decreased the expression of EP1 and EP2. EP3 expression was not detected in either the absence or the presence of IL-1β. When celecoxib was also present, IL-1β failed to stimulate PGE2 production and EP4 expression, but its stimulatory effect on COX-2 expression and its inhibitory effect on EP1 and EP2 expression were unchanged. IL-1β increases the production of PGE2, COX-2, and the PG receptor EP4 in cultured human chondrocytes. The increase in EP4 expression appears to be a result of the increased PGE2 production.

Vaspin Attenuates RANKL-Induced Osteoclast Formation in RAW264.7 Cells

Visceral adipose tissue-derived serine protease inhibitor (vaspin), an adipokine that was recently identified in a rat model of type 2 diabetes, has been suggested to have an insulin-sensitizing effect. In this study, we investigated whether vaspin inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis using two types of osteoclast precursors: RAW264.7 cells and bone marrow cells (BMCs). Vaspin inhibited RANKL-induced osteoclastogenesis in RAW264.7 cells and BMCs. Interestingly, vaspin also inhibited the RANKL-induced expression of nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells and BMCs. Furthermore, it inhibited the RANKL-induced upregulation of matrix metalloproteinase-9 and cathepsin K in RAW264.7 cells. Thus, we suggest that vaspin downregulates osteoclastogenesis in part by inhibiting expression of the transcription factor NFATc1.