Compression and tension variably alter Osteoprotegerin expression via miR-3198 in periodontal ligament cells.

BACKGROUND: Osteoclasts play a critical role in bone resorption due to orthodontic tooth movement (OTM). In OTM, a force is exerted on the tooth, creating compression of the periodontal ligament (PDL) on one side of the tooth, and tension on the other side. In response to these mechanical stresses, the balance of receptor activator of nuclear-factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) shifts to stimulate osteoclastogenesis. However, the mechanism of OPG expression in PDL cells under different mechanical stresses remains unclear. We hypothesized that compression and tension induce different microRNA (miRNA) expression profiles, which account for the difference in OPG expression in PDL cells. To study miRNA expression profiles resulting from OTM, compression force (2 g/cm2) or tension force (15% elongation) was applied to immortalized human PDL (HPL) cells for 24 h, and miRNA extracted. The miRNA expression in each sample was analyzed using a human miRNA microarray, and the changes of miRNA expression were confirmed by real-time RT-PCR. In addition, miR-3198 mimic and inhibitor were transfected into HPL cells, and OPG expression and production assessed. RESULTS: We found that certain miRNAs were expressed differentially under compression and tension. For instance, we observed that miR-572, - 663, - 575, - 3679-5p, UL70-3p, and - 3198 were upregulated only by compression. Real-time RT-PCR confirmed that compression induced miR-3198 expression, but tension reduced it, in HPL cells. Consistent with previous reports, OPG expression was reduced by compression and induced by tension, though RANKL was induced by both compression and tension. OPG expression was upregulated by miR-3198 inhibitor, and was reduced by miR-3198 mimic, in HPL cells. We observed that miR-3198 inhibitor rescued the compression-mediated downregulation of OPG. On the other hand, miR-3198 mimic reduced OPG expression under tension. However, RANKL expression was not affected by miR-3198 inhibitor or mimic. CONCLUSIONS: We conclude that miR-3198 is upregulated by compression and is downregulated by tension, suggesting that miR-3198 downregulates OPG expression in response to mechanical stress.

Nutritional supplementation with myo-inositol in growing mice specifically augments mandibular endochondral growth.

INTRODUCTION: The purpose of this study was to examine growth-promoting effects of myo-inositol nutritional supplementation on the mandible in experimental animals. METHODS: Mice were fed on diets that contained various concentration of myo-inositol for 3 to 12 weeks. The length of the mandible, maxilla, and femur were measured on µCT images. The mandible and tibia were examined histologically and immunohistochemically. The effects of myo-inositol on cell proliferation and chondrocytic differentiation were examined using ATDC5 cells. RESULTS: Myo-inositol supplementation had no effects on body weight, length, and maxilla and femur lengths. However, the length of mandible and the thickness of the mandibular condylar cartilage (MCC) were increased by myo-inositol supplement. Microarray analysis revealed that Pik3cd was highly expressed in MCC as compared to that in the cartilage of the tibial growth plate, which was confirmed by real-time RT-PCR and immunohistochemistry. ATDC5 cells also highly expressed Pik3CD. Myoinositol induced increases in cell proliferation and chondrocytic differentiation in ATDC5 cells. The addition of a PIK3CD inhibitor blocked the induction of cell proliferation by myo-inositol in ATDC5 cells. CONCLUSIONS: Nutritional supplementation with myo-inositol in growing mice augmented mandibular endochondral growth without any systemic effects. The specific promotion of mandibular growth by myoinositol is primarily dependent on the specific intensive expression of PIK3CD in the MCC.

Single Local Injection of Epigallocatechin Gallate-Modified Gelatin Attenuates Bone Resorption and Orthodontic Tooth Movement in Mice.

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.

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-ß has been implicated in the growth of articular cartilage including MCC. Recently, Asporin, decoy to TGF-ß, was discovered and it blocks TGF-ß 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-ß 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-ß 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.

Pathways that Regulate ROS Scavenging Enzymes, and Their Role in Defense Against Tissue Destruction in Periodontitis.

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.

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.