Periodontitis, an inflammatory disease that affects the tissues surrounding the teeth, is a common disease worldwide. It is caused by a dysregulation of the host inflammatory response to bacterial infection, which leads to soft and hard tissue destruction. In particular, it is the excessive inflammation in response to bacterial plaque that leads to the release of reactive oxygen species (ROS) from neutrophils, which, then play a critical role in the destruction of periodontal tissue. Generally, ROS produced from immune cells exhibit an anti-bacterial effect and play a role in host defense and immune regulation. Excessive ROS, however, can exert cytotoxic effects, cause oxidative damage to proteins, and DNA, can interfere with cell growth and cell cycle progression, and induce apoptosis of gingival fibroblasts. Collectively, these effects enable ROS to directly induce periodontal tissue damage. Some ROS also act as intracellular signaling molecules during osteoclastogenesis, and can thus also play an indirect role in bone destruction. Cells have several protective mechanisms to manage such oxidative stress, most of which involve production of cytoprotective enzymes that scavenge ROS. These enzymes are transcriptionally regulated via NRF2, Sirtuin, and FOXO. Some reports indicate an association between periodontitis and these cytoprotective enzymes' regulatory axes, with superoxide dismutase (SOD) the most extensively investigated. In this review article, we discuss the role of oxidative stress in the tissue destruction manifest in periodontitis, and the mechanisms that protect against this oxidative stress.

It has been reported that reactive oxygen species (ROS), such as hydrogen peroxide and superoxide, take part in osteoclast differentiation as intra-cellular signaling molecules. The current assumed signaling cascade from RANK to ROS production is RANK, TRAF6, Rac1, and then Nox. The target molecules of ROS in RANKL signaling remain unclear; however, several reports support the theory that NF-κB signaling could be the crucial downstream signaling molecule of RANKL-mediated ROS signaling. Furthermore, ROS exert cytotoxic effects such as peroxidation of lipids and phospholipids and oxidative damage to proteins and DNA. Therefore, cells have several protective mechanisms against oxidative stressors that mainly induce cytoprotective enzymes and ROS scavenging. Three well-known mechanisms regulate cytoprotective enzymes including Nrf2-, FOXO-, and sirtuin-dependent mechanisms. Several reports have indicated a crosslink between FOXO- and sirtuin-dependent regulatory mechanisms. The agonists against the regulatory mechanisms are reported to induce these cytoprotective enzymes successfully. Some of them inhibit osteoclast differentiation and bone destruction via attenuation of intracellular ROS signaling. In this review article, we discuss the above topics and summarize the current information available on the relationship between cytoprotective enzymes and osteoclastogenesis.

Reactive oxygen species (ROS) play a role in intracellular signaling during osteoclastogenesis. We previously reported that transcriptional factor nuclear factor E2-related factor 2 (Nrf2) was exported from the nucleus to the cytoplasm by receptor activator of nuclear factor-κB ligand (RANKL), and that Nrf2 negatively regulated osteoclastogenesis via antioxidant enzyme up-regulation. Knockout mice of BTB and CNC homology 1 (Bach1)-the competitor for Nrf2 in transcriptional regulation-was known to attenuate RANKL-mediated osteoclastogenesis, although the mechanism remains unclear. Therefore, we hypothesized that RANKL could be involved in the nuclear translocation of Bach1, which would attenuate Nrf2-mediated antioxidant enzymes, thereby augmenting intracellular ROS signaling in osteoclasts. RANKL induced Bach1 nuclear import and Nrf2 nuclear export. Induction of Bach1 nuclear export increased Nrf2 nuclear import, augmented antioxidant enzyme expression, and, thus, diminished RANKL-mediated osteoclastogenesis via attenuated intracellular ROS signaling. Finally, an in vivo mouse bone destruction model clearly demonstrated that induction of Bach1 nuclear export inhibited bone destruction. In this study, we report that RANKL favors osteoclastogenesis via attenuation of Nrf2-mediated antioxidant enzyme expression by competing with Bach1 nuclear accumulation. Of importance, induction of Bach1 nuclear export activates Nrf2-dependent antioxidant enzyme expression, thereby attenuating osteoclastogenesis. Bach1 nuclear export might be a therapeutic target for such bone destructive diseases as rheumatoid arthritis, osteoporosis, and periodontitis.-Kanzaki, H., Shinohara, F., Itohiya, K., Yamaguchi, Y., Katsumata, Y., Matsuzawa, M., Fukaya, S., Miyamoto, Y., Wada, S., Nakamura, Y. RANKL induces Bach1 nuclear import and attenuates Nrf2-mediated antioxidant enzymes, thereby augmenting intracellular reactive oxygen species signaling and osteoclastogenesis in mice.

Bone destructive diseases are common worldwide and are caused by dysregulation of osteoclast formation and activation. During osteoclastogenesis, reactive oxygen species (ROS) play a role in the intracellular signalling triggered by receptor activator of nuclear factor-jB ligand (RANKL) stimulation. Previously, we demonstrated that induction of antioxidant enzymes by Nrf2 activation using Nrf2-gene transfer, an ETGEpeptide or polyphenols, successfully ameliorated RANKL-dependent osteoclastogenesis. Dimethyl fumarate (DMF) has been shown to activate Nrf2 signalling and has been lately used in clinical trials for neurodegenerative diseases. In this study, we hypothesized that Nrf2 activation by DMF would inhibit osteoclastogenesis and bone destruction via attenuation of intracellular ROS signalling through antioxidant mechanisms. RAW 264.7 cells were used as osteoclast progenitor cells. We found that DMF induced Nrf2 translocation to the nucleus, augmented Nrf2 promoter-luciferase reporter activity and increased antioxidant enzyme expression. Using flow cytometry, we found that DMF attenuated RANKL-mediated intracellular ROS generation, which resulted in the inhibition of RANKL-mediated osteoclastogenesis. Local DMF injection into the calvaria of male BALB/c mice resulted in attenuated bone destruction in lipopolysaccharide-treated mice. In conclusion, we demonstrated in a preclinical setting that DMF inhibited RANKL-mediated osteoclastogenesis and bone destruction via induction of Nrf2-mediated transcription of antioxidant genes and consequent decrease in intracellular ROS levels. Our results suggest that DMF may be a promising inhibitor of bone destruction in diseases like periodontitis, rheumatoid arthritis and osteoporosis.Keywords: osteoclast Nrf2 dimethyl fumarate receptor activator of nuclear factor-kB ligand oxidative stress reactive oxygen species antioxidant response HO-1

Osteoclastic bone resorption enables orthodontic tooth movement (OTM) in orthodontic treatment. Previously, we demonstrated that local epigallocatechin gallate (EGCG) injection successfully slowed the rate of OTM; however, repeat injections were required. In the present study, we produced a liquid form of EGCG-modified gelatin (EGCG-GL) and examined the properties of EGCG-GL with respect to prolonging EGCG release, NF-E2-related factor 2 (Nrf2) activation, osteoclastogenesis inhibition, bone destruction, and OTM. We found EGCG-GL both prolonged the release of EGCG and induced the expression of antioxidant enzyme genes, such as heme oxygenase 1 (Hmox1) and glutamate-cysteine ligase (Gclc), in the mouse macrophage cell line, RAW264.7. EGCG-GL attenuated intracellular reactive oxygen species (ROS) levels were induced by the receptor activator of nuclear factor-kB ligand (RANKL) and inhibited RANKL-mediated osteoclastogenesis in vitro. An animal model of bone destruction, induced by repeat Lipopolysaccharide (LPS)-injections into the calvaria of male BALB/c mice, revealed that a single injection of EGCG-GL on day-1 could successfully inhibit LPS-mediated bone destruction. Additionally, experimental OTM of maxillary first molars in male mice was attenuated by a single EGCG-GL injection on day-1. In conclusion, EGCG-GL prolongs the release of EGCG and inhibits osteoclastogenesis via the attenuation of intracellular ROS signaling through the increased expression of antioxidant enzymes. These results indicate EGCG-GL would be a beneficial therapeutic approach both in destructive bone disease and in controlling alveolar bone metabolism.

Zn3N2 has been reported to have high electron mobility even in polycrystalline films. The high mobility in polycrystalline films is a striking feature as compared with group-III nitrides. However, the origins of the high mobility have not been elucidated to date. In this paper, we discuss the reason for high mobility in Zn3N2. We grew epitaxial and polycrystalline films of Zn3N2. Electron effective mass (m*) was determined optically and found to decrease with a decrease in electron density. Using a nonparabolic conduction band model, the m* at the bottom of the conduction band was derived to be (0.08 ± 0.03)m0 (m0 denotes the free electron mass), which is comparable to that in InN. Optically determined intra-grain mobility (μopt) in the polycrystalline films was higher than 110 cm2 V−1 s−1, resulting from the small m*. The Hall mobility (μH) in the polycrystalline films was significantly smaller than μopt, indicating that electron transport is impeded by scattering at the grain boundaries. Nevertheless, μH higher than 70 cm2 V−1 s−1 was achievable owing to the beneficial effect of the high μopt. As for the epitaxial films, we revealed that electron transport is hardly affected by grain boundary scattering and is governed solely by ionized impurity scattering. The findings in this study suggest that Zn3N2 is a high-mobility semiconductor with small effective mass.

A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure in which mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions (PAJs) and wrap around a multiply-branched spine, forming synaptic junctions. Here, we electron microscopically analyzed the ultrastructure of this synapse in afadin-deficient mice. Transmission electron microscopy analysis revealed that typical PAJs with prominent symmetrical plasma membrane darkening undercoated with the thick filamentous cytoskeleton were observed in the control synapse, whereas in the afadin-deficient synapse, atypical PAJs with the symmetrical plasma membrane darkening, which was much less in thickness and darkness than those of the control typical PAJs, were observed. Immunoelectron microscopy analysis revealed that nectin-1, nectin-3, and N-cadherin were localized at the control typical PAJs, whereas nectin-1 and nectin-3 were localized at the afadin-deficient atypical PAJs to extents lower than those in the control synapse and N-cadherin was localized at their nonjunctional flanking regions. These results indicate that the atypical PAJs are formed by nectin-1 and nectin-3 independently of afadin and N-cadherin and that the typical PAJs are formed by afadin and N-cadherin cooperatively with nectin-1 and nectin-3. Serial block face-scanning electron microscopy analysis revealed that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities, and the density of synaptic vesicles docked to active zones were decreased in the afadin-deficient synapse. These results indicate that afadin plays multiple roles in the complex ultrastructural morphogenesis of hippocampal mossy fiber synapses.

Cu3N is a semiconductor with a bandgap of ∼1.4 eV, which is ideal for solar energy conversion applications. To obtain high-efficiency Cu3N-based solar cells, the fabrication of p-n homojunctions is desirable. Studies on the bipolar doping of Cu3N have been initiated very recently. In this study, we demonstrate that lithium-insertion into p-type Cu3N films using a soft chemical treatment causes a p- to n-type conversion and nonmetal–metal transition. This lithium insertion was achieved by treating p-type Cu3N films with n-butyllithium solutions at moderate temperatures of 313–343 K, such that the lithium atoms diffused into Cu3N and occupied the vacant interstitial sites of the crystalline lattice. As a result, the lithium-inserted Cu3N (Li x Cu3N) films became n-type semiconductors, indicating that the lithium atoms act as electron donors. Moreover, Li x Cu3N films with x > 0.05 were found to show metallic electrical conductivity with resistivities of (2–4) × 10–3 Ω cm. This metallic behavior was also observed in the optical transmittance and reflectance data. The findings of this study allowed us to prepare p-type, n-type, and metallic Cu3N-based films. These results may pave the way for the realization of nontoxic wholly Cu3N-based homojunction solar cells delivering high performance.