Kaoru Inami

Augmentation of TNF-induced osteoclast differentiation by inhibition of ERK and activation of p38: Similar intracellular signaling between RANKL- and TNF-induced osteoclast differentiation

Abstract Research in osteoclast differentiation has been greatly advanced since the identification of receptor activator of nuclear factor-κB ligand (RANKL) as osteoclast differentiation factor. The mechanisms of RANKL-induced osteoclast differentiation have been extensively investigated. Mitogen-activated protein kinases (MAPKs) were shown to play crucial roles in RANKL-induced osteoclast differentiation. RANKL-induced osteoclast differentiation was enhanced by inhibition of extracellular signal-regulated kinase (ERK), whereas it was suppressed by inhibition of p38 MAPK. It was reported that tumor necrosis factor (TNF), a major proinflammatory cytokine, induced osteoclast differentiation independently of RANKL. A report showed that inhibition of p38 suppressed TNF-induced osteoclast differentiation, whereas inhibition of ERK did not augment TNF-induced osteoclast differentiation. In this study we reevaluated the roles for MAPKs in TNF-induced osteoclast differentiation. In contrast with the previous report, pretreatment of mouse monocytic RAW264 cells with MAPK/ERK kinase (MEK) inhibitors including PD98059 and U-0126 augmented TNF-induced osteoclast differentiation. Furthermore, we found that U-0126 was more effective in augmentation of osteoclast differentiation than PD98059. Western blot analysis showed that U-0126 inhibited ERK phosphorylation and enhanced p38 phosphorylation, whereas PD98059 inhibited both ERK and p38 phosphorylation. SB203580, a p38 inhibitor, suppressed TNF-induced osteoclast differentiation, and inhibited p38 phosphorylation whereas it augmented ERK phosphorylation. These results demonstrate that ERK inhibition and p38 activation play crucial roles in both RANKL- and TNF-induced osteoclast differentiation.

Interferon-γ enhances the efficacy of autogenous bone grafts by inhibiting postoperative bone resorption in rat calvarial defects

PURPOSEnInterferon (IFN)-γ is a major cytokine produced by immune cells that plays diverse roles in modulating both the immune system and bone metabolism, but its role in autogenous bone grafting remains unknown. Here, we present that local IFN-γ administration improved the efficacy of autogenous bone graft treatment in an experimental rat model.nnnMETHODSnAn autogenous bone graft model was prepared with critically sized rat calvariae defects. Four weeks (w) after bone graft implantation, rats were treated locally with IFN-γ or were not treated. The effect of IFN-γ on bone formation was evaluated for up to 8w with micro-computed tomography, quantitative histomorphometry, and Von Kossa staining. Osteoclastogenesis was assessed by tartrate-resistant acid phosphatase staining. Immunohistochemistry staining or quantitative polymerase chain reactions were used to estimate the expression of osteoclast differentiation factor and inflammatory cytokines including tumor necrosis factor (TNF)-α, a well-known stimulant of osteoclastogenesis and an inhibitor of osteoblast activity, in defects.nnnRESULTSnNewly formed bone gradually replaced the autogenous bone grafts within 4w, although severe bone resorption with osteoclastogenesis and TNF-α expression occurred after 6w in the absence of IFN-γ administration. IFN-γ administration markedly attenuated bone loss, osteoclastogenesis, and TNF-α expression, while it enhanced bone formation at 8w.nnnCONCLUSIONnLocal IFN-γ administration promoted bone formation in autogenous bone grafts possibly via regulating osteoclastogenesis and TNF-α expression. The data provide insights into the potential roles of IFN-γ in autogenous bone grafting.

Direct effects of estrogen on differentiation and apoptosis of osteoclasts

Abstract Osteoclasts play a crucial role in bone resorption. Since osteoclast differentiation/activation is involved in orthodontic tooth movement at the compression sites, the investigation on osteoclasts is very important to the field of orthodontics. It is well known that estrogen has the protective effect on bone. However, the mechanisms by which estrogen prevents bone loss remain to be elucidated. Although estrogen was recently reported to induce apoptosis of osteoclasts, the precise mechanisms of estrogen-induced osteoclast apoptosis remained controversial with regard to whether estrogen affects osteoclasts directly or not. Here we investigated whether estrogen directly induces differentiation and apoptosis of osteoclasts in vitro using mouse monocytic RAW264 cells differentiated into osteoclasts by RANKL. It was observed that estrogen inhibited RANKL-induced osteoclast differentiation of RAW264 cells in a dose-dependent manner. Estrogen suppressed p38 phosphorylation while it enhanced ERK phosphorylation induced by RANKL, suggesting that modulation of MAPK signaling may be involved in inhibition of osteoclast differentiation by estrogen. Next, it was shown that estrogen dose-dependently augmented caspase-3 activation in osteoclasts differentiated from RAW264 cells by RANKL, demonstrating that estrogen directly enhanced apoptosis of osteoclasts. Estrogen-induced caspase-3 activation was attenuated by ICI 182,780, suggesting that the effects of estrogen on osteoclast apoptosis is mediated through estrogen receptors. Thus, these results suggest that estrogen may directly inhibit differentiation and induce apoptosis of osteoclasts.

Cell survival and gene expression under compressive stress in a three-dimensional in vitro human periodontal ligament-like tissue model.

This study investigated cell survival and gene expression under various compressive stress conditions mimicking orthodontic force by using a newly developed in vitro model of human periodontal ligament-like tissue (HPdLLT). The HPdLLT was developed by three-dimensional culturing of human periodontal ligament fibroblasts in a porous poly-l-lactide matrix with threefold increased culture media permeability due to hydrophilic modification. In vitro HPdLLTs in experimental groups were subjected to 5, 15, 25 and 35xa0g/cm2 compressive stress for 1, 3, 7 or 14xa0days; controls were cultured over the same periods without compressive stress. Cell morphology and cell apoptosis in the experimental and control groups were investigated using scanning electron microscopy and caspase-3/7 detection. Real-time polymerase chain reaction was performed for seven osteogenic and osteoclastic genes. Similar extracellular matrix and spindle-shaped cells were observed inside or on the surface of in vitro HPdLLTs, with no relation to compressive stress duration or intensity. Similar caspase-3/7 activity indicating comparable apoptosis levels was observed in all samples. Receptor activator of nuclear factor kappa-B ligand and bone morphogenetic protein 2 genes showed characteristic “double-peak” expression at 15 and 35xa0g/cm2 on day 14, and alkaline phosphatase and periodontal ligament-associated protein 1 expression peaked at 5xa0g/cm2 on day 14; other genes also showed time-dependent and load-dependent expression patterns. The in vitro HPdLLT model system effectively mimicked the reaction and gene expression of the human periodontal ligament in response to orthodontic force. This work provides new information on the effects of compressive stress on human periodontal ligament tissue.

In vitro human periodontal ligament-like tissue formation with porous poly-l-lactide matrix

This study aimed to establish an in vitro human periodontal ligament-like tissue (HPdLLT) by three-dimensional culturing of human periodontal ligament fibroblasts (HPdLFs) in a porous poly-L-lactide (PLLA) matrix modified hydrophilically with ammonia solution. After ammonia modification, the surface roughness and culture-medium-soaking-up ability of the PLLA matrix increased, whereas the contact angle of water drops decreased. The thickness, porosity, and pore size of the PLLA matrix were 400±50 μm, 83.3%, and 75-150 μm, respectively. HPdLFs (1×10(5) cells) were seeded on the modified PLLA matrix and centrifuged to facilitate seeding into its interior and cultured for 14 days. Scanning electron microscope (SEM) observation, proliferation assay, picrosirius-red staining, and real-time polymerase chain reaction (RT-PCR) for type-1 collagen (COL1), periodontal ligament associated protein-1 (PLAP-1), fibroblast growth factor-2 (FGF-2), and alkaline phosphatase (ALP) mRNA were conducted on days 1, 3, 7, and 14. HPdLFs were observed entirely from the surface to the rear side of the matrix. Cell proliferation analysis, SEM observation, and picrosirius-red staining showed both progressive growth of 3D-cultured HPdLFs and extracellular matrix maturation by the secretion of COL1 and type 3 collagen (COL3) from days 1 to 14. Expressions of COL1, PLAP-1, and FGF-2 mRNA suggested the formation of cellular components and supplementation of extracellular components. Expressions of ALP, COL1, and PLAP-1 mRNA suggested the osteogenic potential of the HPdLLT. The results indicated in vitro HPdLLT formation, and it could be used in future periodontal ligament tissue engineering to achieve optimal periodontal regeneration.

Induction of osteoclast differentiation by NOC-18, a long-acting nitric oxide donor

Abstract It is well established that osteoclast differentiation/activation is involved in orthodontic tooth movement at the compression sites. However, the mechanism of osteoclast differentiation/activation in orthodontic tooth movement remains unclear. Nitric oxide (NO) has been reported to play a crucial role in bone remodeling induced by mechanical loading, and several reports showed that inhibition of NO synthases prevented orthodontic tooth movement in animal models. Here, we investigated whether osteoclast differentiation can be induced by NO in vitro. Osteoclast differentiation was slightly induced by NOC-18, a long-acting NO donor, and RANKL-induced osteoclast differentiation was further augmented by NOC-18. Short-acting NO donors including NOC-15, NOC-9 and sodium nitroprusside (SNP) did not significantly induce osteoclast differentiation. Since it has been reported that mitogen-activated protein kinase (MAPK) signaling plays a crucial role in RANKL-induced osteoclast differentiation and RANKL-induced osteoclast differentiation was enhanced by inhibition of extracellular signal-regulated kinase (ERK) whereas it was suppressed by inhibition of p38 MAPK, involvement of MAPK signaling in stimulation of osteoclast differentiation by NOC-18 was investigated. Western blot analysis demonstrated that pretreatment with NOC-18 inhibited RANKL-induced phosphorylation of ERK whereas it augmented RANKL-induced phosphorylation of p38 MAPK. Furthermore, mouse M-CSF-dependent bone marrow macrophages were induced to differentiate into osteoclasts by NOC-18. These results suggest that NOC-18 enhanced osteoclast differentiation via MAPK signaling in accordance with RANKL-induced osteoclast differentiation.

The increased ratio of 11β-hydroxysteroid dehydrogenase type 1 versus 11β-hydroxysteroid dehydrogenase type 2 in chronic periodontitis irrespective of obesity

Abstract11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts inactive cortisone to active cortisol, has been reported to play an important role in metabolic diseases as well as chronic inflammatory diseases. The involvement of 11β-HSD1 in chronic periodontitis was investigated in the present study. The relationship between the levels of 11β-HSD1, chronic periodontitis, and body mass index (BMI) was analyzed. The expression of 11β-HSD1 mRNA was significantly higher in the chronic periodontitis group than in the control group. Since the expression of 11β-HSD2, which converts active cortisol to inactive cortisone, was slightly lower in the chronic periodontitis group than in the controls, the ratio of 11β-HSD1 versus 11β-HSD2 was significantly higher in the chronic periodontitis group than in the controls. A correlation was not observed between BMI and the level of 11β-HSD1 or between BMI and the ratio of 11β-HSD1 versus 11β-HSD2. These results suggested that an increase in the ratio of 11β-HSD1 versus 11β-HSD2 was associated with chronic periodontitis irrespective of obesity.

An inhibitory role for caspase-3 at the late stage of RANKL-induced osteoclast differentiation in RAW264 cells and mouse bone marrow macrophages.

Osteoclast differentiation/activation is involved in orthodontic tooth movement at the compression sites of the alveolar bone. RANKL, a member of the TNF family expressed in osteoblasts, binds to RANK, a member of the TNF receptor family expressed on preosteoclasts, resulting in differentiation of preosteoclasts into mature osteoclasts. Several members of the TNF family, such as TNF and Fas ligand, can induce apoptosis by activation of caspase‐3. We have investigated whether caspase‐3 be involved in the late stage of RANKL‐induced osteoclast differentiation. Increased active caspase‐3 was found in mouse monocytic RAW264 cells differentiated into mature osteoclasts by treatment with RANKL for 3 days. Co‐treatment with Z‐Asp‐CH2‐DCB, a caspase‐3‐specific inhibitor, augmented RANKL‐induced osteoclast differentiation in RAW264 cells, also seen in mouse bone marrow macrophages. This suggests that activation of caspase‐3 may play an inhibitory role at the late stage of RANKL‐induced osteoclast differentiation.

Facilitation of experimental tooth movement by NOC-18, a long-acting nitric oxide donor

Abstract Purpose In orthodontic tooth movement, osteoclasts play a crucial role in bone resorption on the compression side of the alveolar bone. It has been reported that nitric oxide is involved in bone remodeling caused by mechanical loading, and we previously reported that NOC-18, a long-acting nitric oxide donor, augmented RANKL-induced osteoclast differentiation in mouse monocytic RAW264 cells as well as mouse bone marrow macrophages. In this study, we investigated whether NOC-18 facilitated experimental tooth movement in mice. Materials and methods Eight-week-old male ddY mice were used. Experimental tooth movement was induced by the insertion of an orthodontic elastic between the left maxillary first molar and second molar. Just after the insertion of the elastic, NOC-18 was intraperitoneally administered once, and mice were killed after 3 days. For the detection of osteoclasts, HE staining, TRAP staining, and immunostaining for cathepsin K were performed. Results An intraperitoneal injection of NOC-18 significantly increased the distance of experimental tooth movement. Furthermore, the number and area of osteoclasts on the compression side of the alveolar bone surface was significantly higher in the NOC-18 group. Conclusion These results suggested that systemic administration of NOC-18 might have some effect to facilitate experimental tooth movement in mice via the augmentation of osteoclast differentiation on the compression side of the alveolar bone.