Yutaka Miyamoto

RANKL induces Bach1 nuclear import and attenuates Nrf2-mediated antioxidant enzymes, thereby augmenting intracellular reactive oxygen species signaling and osteoclastogenesis in mice

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. FASEB J. 31, 781–792 (2017). http://www.fasebj.org

Molecular regulatory mechanisms of osteoclastogenesis through cytoprotective enzymes

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.

A-Disintegrin and Metalloproteinase (ADAM) 17 Enzymatically Degrades Interferon-gamma.

Interferon-gamma (IFN-γ) is a pleiotropic cytokine that exerts anti-tumor and anti-osteoclastogenic effects. Although transcriptional and post-transcriptional regulation of IFN-γ is well understood, subsequent modifications of secreted IFN-γ are not fully elucidated. Previous research indicates that some cancer cells escape immune surveillance and metastasize into bone tissue by inducing osteoclastic bone resorption. Peptidases of the a-disintegrin and metalloproteinase (ADAM) family are implicated in cancer cell proliferation and tumor progression. We hypothesized that the ADAM enzymes expressed by cancer cells degrades IFN-γ and attenuates IFN-γ-mediated anti-tumorigenic and anti-osteoclastogenic effects. Recombinant ADAM17 degraded IFN-γ into small fragments. The addition of ADAM17 to the culture supernatant of stimulated mouse splenocytes decreased IFN-γ concentration. However, ADAM17 inhibition in the stimulated mouse T-cells prevented IFN-γ degradation. ADAM17-expressing human breast cancer cell lines MCF-7 and MDA-MB-453 also degraded recombinant IFN-γ, but this was attenuated by ADAM17 inhibition. Degraded IFN-γ lost the functionality including the inhibititory effect on osteoclastogenesis. This is the first study to demonstrate the extracellular proteolytic degradation of IFN-γ by ADAM17. These results suggest that ADAM17-mediated degradation of IFN-γ may block the anti-tumorigenic and anti-osteoclastogenic effects of IFN-γ. ADAM17 inhibition may be useful for the treatment of attenuated cancer immune surveillance and/or bone metastases.

Dimethyl fumarate inhibits osteoclasts via attenuation of reactive oxygen species signalling by augmented antioxidation

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‐κB ligand (RANKL) stimulation. Previously, we demonstrated that induction of antioxidant enzymes by Nrf2 activation using Nrf2‐gene transfer, an ETGE‐peptide 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.

Asporin stably expressed in the surface layer of mandibular condylar cartilage and augmented in the deeper layer with age

Mandibular condylar cartilage (MCC) exhibits dual roles both articular cartilage and growth center. Of many growth factors, TGF-β has been implicated in the growth of articular cartilage including MCC. Recently, Asporin, decoy to TGF-β, was discovered and it blocks TGF-β signaling. Asporin is expressed in a variety of tissues including osteoarthritic articular cartilage, though there was no report of Asporin expression in MCC. In the present study, we investigated the temporal and spatial expression of Asporin in MCC. Gene expression profile of MCC and epiphyseal cartilage in tibia of 5 weeks old ICR mice were firstly compared with microarray analysis using the laser capture microdissected samples. Variance of gene expression was further confirmed by real-time RT-PCR and immunohistochemical staining at 1,3,10, and 20 weeks old. TGF-β and its signaling molecule, phosphorylated Smad-2/3 (p-Smad2/3), were also examined by immunohistochemical staining. Microarray analysis revealed that Asporin was highly expressed in MCC. Real-time RT-PCR analysis confirmed that the fibrous layer of MCC exhibited stable higher Asporin expression at any time points as compared to epiphyseal cartilage. This was also observed in immunohistochemical staining. Deeper layer in MCC augmented Asporin expression with age. Whereas, TGF-β was stably highly observed in the layer. The fibrous layer of MCC exhibited weak staining of p-Smad2/3, though the proliferating layer of MCC was strongly stained as compared to epiphyseal cartilage of tibia at early time point. Consistent with the increase of Asporin expression in the deeper layer of MCC, the intensity of p-Smad-2/3 staining was decreased with age. In conclusion, we discovered that Asporin was stably expressed at the fibrous layer of MCC, which makes it possible to manage both articular cartilage and growth center at the same time.

Possible alternative treatment for mandibular asymmetry by local unilateral IGF-1 injection into the mandibular condylar cavity: Experimental study in mice

Introduction: The purpose of this study was to investigate whether a local unilateral IGF‐1 injection into the mandibular condylar cavity can induce unilateral endochondral mandibular growth without any systemic adverse effects. Methods: Seventy‐five 3‐week‐old male Jcl:ICR mice were used in this study. The mice were divided into 2 groups: control group (n = 22) and IGF‐1 group (n = 53). In the IGF‐1 group, human IGF‐1 was injected into the right mandibular condylar cavity, and phosphate‐buffered saline solution was injected into the left cavity, 3 times per week for 10 weeks. Results: There was no significant difference in body weight, serum human IGF‐1 concentration, and soft tissue thickness of the cheeks including the masseter muscles between the 2 groups. Unilateral IGF‐1 injection induced a lateral shift of the mandible to the contralateral side, and microcomputed tomogtraphy analysis showed that unilateral IGF‐1 injection induced endochondral growth in the condyle. Col2, Ihh, and Runx2 were extensively upregulated by the local unilateral IGF‐1 injection in real‐time reverse transcription polymerase chain reaction analysis. Proliferation marker KI67, IGF‐1 signaling molecule AKT1, and chondrogenic differentiation marker Col2 were strongly expressed in the IGF‐1 injected condyle by immunohistochemistry. Vital labeling showed that the distance between the labels was increased in the IGF‐1 injection group compared with that of the control group. Conclusions: The results verified in this study indicated that local unilateral IGF‐1 injection into the mandibular condylar cavity successfully induced unilateral endochondral mandibular growth in mice without any systemic adverse effects. Thus, local unilateral IGF‐1 injection into the mandibular condylar cavity could be a useful alternative for mandibular asymmetry therapy during the growth period. However, additional experimental and clinical studies will be necessary to prove the real effect of this new therapy. HighlightsIGF‐1 was injected unilaterally into the mandibular condylar cavity in mice.Local IGF‐1 injection induced a lateral shift of the mandible.Local injection into the condylar cavity induced endochondral growth and ossification.Chondrogenic and osteoblastic gene expression was upregulated.No systemic effects were found.

Orthodontic treatment of acquired open bite accompanied with extreme mesially inclined mandibular molars

Acquired open bite mainly occurs after adolescence and is caused by a pathological or biological process. We herein report the case of an 18-year and 10-month-old Japanese woman. Her chief complaint was difficulty with mastication. Only distal cusps of first molars and second molars had contact. An angle class II molar relationship and extreme mesially inclined mandibular first molars were observed. The degree of overjet was +6.1mm and overbite was -2.0mm. There were no symptoms or abnormal findings in temporomandibular joints by radiography and magnetic resonance imaging. A skeletal class II jaw relationship was indicated. Therefore, the patient was diagnosed with a skeletal class II acquired open bite malocclusion accompanied with extreme mesial inclination of mandibular first molars. Orthodontic treatment with extraction of maxillary first premolars was performed. After stabilizing the maxillary dental arch, mandibular molars were uprighted and intruded by class III elastics. After treatment, overjet was +3.0mm and overbite was +2.3mm. Occlusal cusp-fossa relationships were established and canine relationships were class I. The lower occlusal plane and mandible were rotated counterclockwise, which helped to achieve proper overbite. After 2 years of retention, occlusion was well maintained.

Occlusal hypofunction mediates alveolar bone apposition via relative augmentation of TGF-βsignaling by decreased Asporin production in rats

The periodontal ligament (PDL) maintains alveolar bone homeostasis against mastication force. Occlusal hypofunction, which lost mastication force, narrows the PDL by alveolar bone apposition, though the mechanisms remain unclear. Asporin is a secreted extracellular matrix protein and preferentially expressed in the PDL. Asporin binds directly with transforming growth factor-beta (TGF-β) and inhibits TGF-β/Smad signaling, resulting in the inhibition of bone formation. In the present study, we hypothesized that Asporin is downregulated by occlusal hypofunction, which results in the increased alveolar bone apposition via relative upregulation of TGF-β signaling. To clarify the hypothesis, we employed in vivo experiments using rats. Crowns of lower right first molar in Wistar rats were removed to induce occlusal hypofunction in upper right first molar. Alveolar bone apposition was examined by histomorphometric analysis of Calcein/Xylenol-orange vital staining. Asporin, TGF-β, and it`s signaling molecule, Smad3 expression were examined in mRNA and protein levels. Osteoblastic differentiation of the cell in the PDL under the occlusal hypofunction were observed using Bone sialoprotein (BSP), Osteocalcin and Osteopontin expression as osteoblastic differentiation marker. We discovered that occlusal hypofunction increased the alveolar bone apposition and downregulated Asporin expression in PDL fibroblasts. Furthermore, occlusal hypofunction relative augmented TGF-β signaling in PDL judged by phosphorylated (p)-Smad2/3 immunohistochemical staining, and upregulated osteoblastic differentiation in PDL. In the present study, we firstly reported that occlusal hypofunction mediates alveolar bone apposition via relative augmentation of TGF-β signaling by decrease of Asporin production. Correspondence to: Yoshiki Nakamura, DDS, Ph D, Department of orthodontics, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama, Kanagawa pref., 230-8501, Japan, Tel: +81-45-580-8507, Fax: +8145-573-9599, E-mail: nakamura-ys@tsurumi-u.ac.jp