Bach1 Inhibition Suppresses Osteoclastogenesis via Reduction of the Signaling via Reactive Oxygen Species by Reinforced Antioxidation.

Bone destructive diseases such as periodontitis are common worldwide and are caused by excessive osteoclast formation and activation. Receptor activator of nuclear factor-κB ligand (RANKL) is essential factor for osteoclastogenesis. This triggers reactive oxygen species (ROS), which has a key role in intracellular signaling as well exerting cytotoxicity. Cells have protective mechanisms against ROS, such as nuclear factor E2-related factor 2 (Nrf2), which controls the expression of many antioxidant enzyme genes. Conversely, BTB and CNC homology 1 (Bach1), a competitor for Nrf2, transcriptionally represses the expression of anti-oxidant enzymes. Previously, we demonstrated that RANKL induces Bach1 nuclear import and attenuates the expression of Nrf2-mediated antioxidant enzymes, thereby augmenting intracellular ROS signaling and osteoclastogenesis. However, it remains unknown if Bach1 inhibitors attenuate osteoclastogenesis. In this study, we hypothesized that Bach1 inhibition would exert an anti-osteoclastogenic effects via diminishing of intracellular ROS signaling through augmented antioxidation. We used RAW 264.7 cells as osteoclast progenitor cells. Using flow cytometry, we found that Bach1 inhibitors attenuated RANKL-mediated ROS generation, which resulted in the inhibition of osteoclastogenesis. Local injection of a Bach1 inhibitor into the calvaria of male BALB/c mice blocked bone destruction induced by lipopolysaccharide. In conclusion, we demonstrate that Bach1 inhibitor attenuates RANKL-mediated osteoclastogenesis and bone destruction in mice by inducing the expression of Nrf2-regulated antioxidant enzymes that consequently decrease intracellular ROS levels. Bach1 inhibitors have potential in inhibiting bone destructive diseases such as periodontitis, rheumatoid arthritis and osteoporosis.

Nrf2 activation in osteoblasts suppresses osteoclastogenesis via inhibiting IL-6 expression.

Bone destructive diseases such as periodontitis and rheumatoid arthritis are caused by excessive activation of osteoclasts. Osteoclastogenesis is regulated by Receptor activator of nuclear factor kappa-ß ligand (RANKL) produced by osteoclastogenesis supporting cells such as osteoblast and osteocyte. Previously, we reported that NF-E2-related factor-2 (Nrf2) activation in osteoclast precursors inhibited osteoclastogenesis and bone destruction via induction of anti-oxidation and thereby attenuated intracellular ROS signaling. However, it still remains unknown whether Nrf2 activation in cells other than osteoclasts give any negative influence on supporting property for osteoclastogenesis. Here we discovered that Nrf2 activation in osteoblasts suppresses indirectly osteoclastogenesis via inhibiting the expression of interleukin-6 (IL-6) which promotes osteoclastogenesis. In this study, 5-aminolevulinic acid hydrochloride (ALA) and sodium ferrous citrate (SFC) was used as the Nrf2 activator. in vitro experiments, using osteoblast cell line, MC3T3-E1, revealed that the expression of IL-6 was increased by LPS stimulation, but decreased after ALA/SFC treatment in mRNA and protein levels. Furthermore, RANKL expression was augmented by LPS, which was blocked by ALA/SFC treatment. Neutralizing antibody against IL-6 confirmed that LPS-mediated RANKL augmentation was dependent on IL-6 induction. in vivo experiments with LPS-mediated bone destruction in mice, confirmed that augmented IL-6 expression in osteoblasts by immunochemical analysis. ALA/SFC treatment attenuated LPS-mediated IL-6 upregulation. These results suggest that Nrf2 activation in osteoblasts suppress IL-6 and inflammatory bone destruction. The Nrf2 activator acts not only on osteoclasts but also on osteoblasts, in other word, Nrf2 activation indirectly suppresses osteoclastogenesis. In conclusion, the Nrf2 activator exhibits dual inhibitory effects via direct action on osteoclast and indirect action on osteoclast supporting cells.

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.

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.

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.

Novel device for application of continuous mechanical tensile strain to mammalian cells.

During orthodontic tooth movement, the periodontal ligament (PDL) is exposed to continuous mechanical strain. However, many researchers have applied cyclic tensile strain, not continuous tensile strain, to PDL cells in vitro because there has been no adequate device to apply continuous tensile strain to cultured cells. In this study, we contrived a novel device designed to apply continuous tensile strain to cells in culture. The continuous tensile strain was applied to human immortalized periodontal ligament cell line (HPL cells) and the cytoskeletal structures of HPL cells were examined by immunohistochemistry. The expression of both inflammatory and osteogenic markers was also examined by real-time reverse transcription polymerase chain reaction. The osteogenic protein, Osteopontin (OPN), was also detected by western blot analysis. The actin filaments of HPL cells showed uniform arrangement under continuous tensile strain. The continuous tensile strain increased the expression of inflammatory genes such as IL-1ß, IL-6, COX-2 and TNF-α, and osteogenic genes such as RUNX2 and OPN in HPL cells. It also elevated the expression of OPN protein in HPL cells. These results suggest that our new simple device is useful for exploring the responses to continuous tensile strain applied to the cells.