Upregulated RANKL Expression Research Articles

An Overview of the Deubiquitinase USP53: A Promising Diagnostic Marker and Therapeutic Target

: Ubiquitination and deubiquitination are important mechanisms to maintain normal physiological activities, and their disorders or imbalances can lead to various diseases. As a subgroup of deubiquitinases (DUBs), the ubiquitin-specific peptidase (USP) family is closely related to many biological processes. USP53, one of the family members, is widely expressed in human tissues and participates in a variety of life activities, such as cell apoptosis, nerve transmission, and bone remodeling. Mutations in the USP53 gene can cause cholestasis and deafness and may also be a potential cause of schizophrenia. Knockout of USP53 can alleviate neuropathic pain induced by chronic constriction injury. Loss of USP53 up-regulates RANKL expression, promotes the cytogenesis and functional activity of osteoclasts, and triggers osteodestructive diseases. USP53 plays a tumor-suppressive role in lung cancer, renal clear cell carcinoma, colorectal cancer, liver cancer, and esophageal cancer but reduces the radiosensitivity of cervical cancer and esophageal cancer to induce radioresistance. Through the in-depth combination of literature and bioinformatics, this review suggested that USP53 may be a good potential biomarker or therapeutic target for diseases.

Exogenous iron caused osteocyte apoptosis, increased RANKL production, and stimulated bone resorption through oxidative stress in a murine model

Iron overload is a risk factor for osteoporosis due to its oxidative toxicity. Previous studies have demonstrated that an excessive amount of iron increases osteocyte apoptosis and receptor activator of nuclear factor κ-B ligand (RANKL) production, which stimulates osteoclast differentiation in vitro. However, the effects of exogenous iron supplementation-induced iron overload on osteocytes in vivo and its role in iron overload-induced bone loss are unknown. This work aimed to develop an iron overloaded murine model of C57BL/6 mice by intraperitoneal administration of iron dextran for two months. The iron levels in various organs, bone, and serum, as well as the microstructure and strength of bone, apoptosis of osteocytes, oxidative stress in bone tissue, and bone formation and resorption, were assessed. The results showed that 2 months of exogenous iron supplementation significantly increased iron levels in the liver, spleen, kidney, bone tissue, and serum. Iron overload negatively affected bone microstructure and strength. Osteocyte apoptosis and empty lacunae rate were elevated by exogenous iron. Iron overload upregulated RANKL expression but had no significant impact on osteoprotegerin (OPG) and sclerostin levels. Static and dynamic histologic analyses and serum biochemical assay showed that iron overload increased bone resorption without significantly affecting bone formation. Exogenous iron promoted oxidative stress in osteocytes in vivo and in vitro. Additional supplementation of iron chelator (deferoxamine) or N-acetyl-l-cysteine (NAC) partially alleviated bone loss, osteocyte apoptosis, osteoclast formation, and oxidative stress due to iron overload. These findings, in line with prior in vitro studies, suggest that exogenous iron supplementation induces osteoclastogenesis and osteoporosis by promoting osteocyte apoptosis and RANKL production via oxidative stress.

Histone demethylase KDM7A regulates bone homeostasis through balancing osteoblast and osteoclast differentiation

Histone methylation plays a crucial role in various cellular processes. We previously reported the in vitro function of histone lysine demethylase 7 A (KDM7A) in osteoblast and adipocyte differentiation. The current study was undertaken to investigate the physiological role of KDM7A in bone homeostasis and elucidate the underlying mechanisms. A conditional strategy was employed to delete the Kdm7a gene specifically in osterix-expressing osteoprogenitor cells in mice. The resulting mutant mice exhibited a significant increase in cancellous bone mass, accompanied by an increase in osteoblasts and bone formation, as well as a reduction in osteoclasts, marrow adipocytes and bone resorption. The bone marrow stromal cells (BMSCs) and calvarial pre-osteoblastic cells derived from the mutant mice exhibited enhanced osteogenic differentiation and suppressed adipogenic differentiation. Additionally, osteoclastic precursor cells from the mutant mice exhibited impaired osteoclast differentiation. Co-culturing BMSCs from the mutant mice with wild-type osteoclast precursor cells resulted in the inhibition of osteoclast differentiation. Mechanistic investigation revealed that KDM7A was able to upregulate the expression of fibroblast activation protein α (FAP) and receptor activator of nuclear factor κB ligand (RANKL) in BMSCs through removing repressive di-methylation marks of H3K9 and H3K27 from Fap and Rankl promoters. Moreover, recombinant FAP attenuated the dysregulation of osteoblast and adipocyte differentiation in BMSCs from Kdm7a deficient mice. Finally, Kdm7a deficiency prevented ovariectomy-induced bone loss in mice. This study establish the role of KDM7A in bone homeostasis through its epigenetic regulation of osteoblast and osteoclast differentiation. Consequently, inhibiting KDM7A may prove beneficial in ameliorating osteoporosis.KDM7A suppresses osteoblast differentiation and bone formation through. upregulating FAP expression and inactivating canonical Wnt signaling, and conversely promotes osteoclast differentiation and bone resorption through upregulating RANKL expression. These are based on its epigenetic removal of the repressive H3K9me2 and H3K27me2 marks from Fap and Rankl promoters. As a result, the expression of KDM7A in osteoprogenitor cells tends to negatively modulate bone mass.

Schnurri-3 inhibition suppresses bone and joint damage in models of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads to systemic and articular bone loss by activating bone resorption and suppressing bone formation. Despite current therapeutic agents, inflammation-induced bone loss in RA continues to be a significant clinical problem due to joint deformity and lack of articular and systemic bone repair. Here, we identify the suppressor of bone formation, Schnurri-3 (SHN3), as a potential target to prevent bone loss in RA. SHN3 expression in osteoblast-lineage cells is induced by proinflammatory cytokines. Germline deletion or conditional deletion of Shn3 in osteoblasts limits articular bone erosion and systemic bone loss in mouse models of RA. Similarly, silencing of SHN3 expression in these RA models using systemic delivery of a bone-targeting recombinant adenoassociated virus protects against inflammation-induced bone loss. In osteoblasts, TNF activates SHN3 via ERK MAPK-mediated phosphorylation and, in turn, phosphorylated SHN3 inhibits WNT/β-catenin signaling and up-regulates RANKL expression. Accordingly, knock-in of a mutation in Shn3 that fails to bind ERK MAPK promotes bone formation in mice overexpressing human TNF due to augmented WNT/β-catenin signaling. Remarkably, Shn3-deficient osteoblasts are not only resistant to TNF-induced suppression of osteogenesis, but also down-regulate osteoclast development. Collectively, these findings demonstrate SHN3 inhibition as a promising approach to limit bone loss and promote bone repair in RA.

Mechanical force regulates root resorption in rats through RANKL and OPG

BackgroundExternal root resorption is one of common complications of orthodontic treatment, while internal root resorption is rarely observed, and the difference between pulp and periodontal tissues during orthodontic treatment is still unknown. The purpose of this study was to evaluate the effects of orthodontic forces on histological and cellular changes of the dental pulp and periodontal tissues.MethodsOrthodontic tooth movement model was established in Forty-eight adult male Wistar rats. The distance of orthodontic tooth movement was quantitatively analyzed. The histological changes of pulp and periodontal tissues were performed by hematoxylin–eosin staining, tartrate-resistant acid phosphate staining was used to analyze the changes of osteoclast number, immunohistochemistry analysis and reverse transcription polymerase chain reaction were used to examine the receptor of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) expression. The width of tertiary dentine was quantitatively analyzed. Tartrate-resistant acid phosphate staining and the erosion area of osteo assay surface plate was used to evaluate osteoclast activity.ResultsThe orthodontic tooth movement distance increased in a force dependent manner, and reached the peak value when orthodontic force is 60 g. Heavy orthodontic force increased the RANKL expression of periodontal ligament srem cells (PDLSCs) which further activated osteoclasts and resulted in external root resorption, while the RANKL expression of dental pulp stem cells (DPSCs) was relatively low to activate osteoclasts and result in internal root resorption, and the dental pulp tend to form tertiary dentine under orthodontic force stimulation.ConclusionsHeavy orthodontic forces activated osteoclasts and triggered external root resorption by upregulating RANKL expression in rat periodontal tissues, while there was no significant change of RANKL expression in dental pulp tissue under heavy orthodontic forces, which prevented osteoclast activation and internal root resorption.

Effects of the Lysine Methyltransferase Inhibitor AZ505 on Bone Metabolism.

BackgroundProtein methylation has important role in regulating diverse cellular responses, including differentiation, by affecting protein activity, stability, and interactions. AZ505 is an inhibitor of the SET and MYND domain-containing protein 2 lysine methylase. In this study, we investigated the effect of AZ505 on osteoblast and osteoclast differentiation in vitro and evaluated the effect of AZ505 in vivo on the long bones in mice.MethodsOsteoblast differentiation was assessed by alkaline phosphatase (ALP) and Alizarin red staining after culturing calvarial preosteoblasts in an osteogenic medium. Osteoclast differentiation was analyzed by tartrate-resistant acid phosphatase (TRAP) staining in bone marrow-derived macrophages cultured with macrophage-colony stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). For in vivo experiments, mice were intraperitoneally injected with AZ505 and femurs were examined by micro-computed tomography.ResultsAZ505 increased ALP and Alizarin red staining in cultured osteoblasts and the expression of osteoblast marker genes, including Runx2 and osteocalcin. AZ505 resulted in decreased TRAP-staining of osteoclasts and expression of c-Fos and nuclear factor of activated T cells transcription factors and osteoclast marker genes, including cathepsin K and dendritic cell-specific transmembrane protein. Unexpectedly, in vivo administration of AZ505 markedly decreased the trabecular bone mass of femurs. In support of this catabolic result, AZ505 strongly upregulated RANKL expression in osteoblasts.ConclusionsThe results indicate that AZ505 has a catabolic effect on bone metabolism in vivo despite its anabolic effect in bone cell cultures. The findings indicate that cell culture data should be extrapolated cautiously to in vivo outcomes for studying bone metabolism.

B-Cell Deficiency Exacerbates Inflammation and Bone Loss in Ligature-Induced Experimental Periodontitis in Mice.

ObjectivePeriodontitis, one of the most prevalent chronic oral infectious diseases in humans, is induced by the breakdown in the balance between the biofilm and host immune system. Previous studies have shown the presence of large numbers of B cells in periodontitis lesions, implicating that B lymphocytes play a predominant role during the pathogenesis of periodontitis. This study aimed to investigate the role of all B cells in the initiation of periodontitis.MethodsExperimental periodontitis was induced in B cell-deficient (CD19Cre) mice and wild-type (WT) control mice by 5-0 silk ligation around the maxillary second molar. Four weeks after ligation, alveolar bone loss was determined by micro-computed tomography. The levels of inflammatory cytokines and receptor activator of NF-κB ligand (RANKL)/osteoprotegerin in periodontal lesions were analyzed using real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. Lymphocyte populations in the cervical lymph nodes and spleen and among the peripheral blood mononuclear cells were detected by flow cytometry.ResultsB-cell deficiency resulted in increased severity of alveolar bone loss in mouse experimental periodontitis, which was associated with increased osteoclast activity and upregulated RANKL expression in the periodontal lesions. In addition, gingiva cytokine expression profiles were shifted to T helper type 1 (Th1) and Th17 in the CD19Cre mice with ligature-induced periodontitis compared with WT mice. In addition, a reduced CD4+/CD8+ T cell ratio was observed in the CD19Cre mice.ConclusionB-cell deficiency exacerbates the inflammation and alveolar bone loss in ligature-induced experimental periodontitis in mice, implicating that B cells may overall play a protective role in the initiation of periodontitis.

Bilirubin and bile acids in osteocytes and bone tissue. Potential role in the cholestatic‐induced osteoporosis

Osteoporosis is a common complication in patients with primary biliary cholangitis. Both bilirubin and lithocholic acid (LCA) result in detrimental effects on osteoblastic cells, and ursodeoxycholic acid (UDCA) counteracts these outcomes. However, there is no information on the consequences of these retained substances of cholestasis and sera from cholestatic patients in osteocytes. The impact of bilirubin, LCA, UDCA and serum from jaundiced patients on viability, differentiation, mineralization and apoptosis has been assessed in MLO-Y4 and MLO-A5 osteocyte cell lines. Effects on gene expression were assessed in these cells and in human bone fragments. Lithocholic acid 10μmol/L and bilirubin 50μmol/L decreased viability in MLO-Y4 and MLO-A5 cells (11% and 53% respectively; P≤.01). UDCA alone or combined with LCA or bilirubin increased cell viability. Jaundiced sera decreased cell viability (56%), an effect which was reverted by UDCA. Bilirubin decreased differentiation by 47% in MLO-Y4 (P≤.01) and mineralization (87%) after 21days in MLO-A5 (P≤.03). Both bilirubin and LCA increased apoptosis in MLO-Y4, and UDCA diminished the apoptotic effect. Moreover, bilirubin down-regulated RUNX2 and up-regulated RANKL gene expression in bone tissue, MLO-Y4 and MLO-A5 cells, and LCA up-regulated RANKL expression in bone tissue. UDCA 100μmol/L increased the gene expression of all these genes in bone tissue and MLO-Y4 cells and neutralized the decreased RUNX2 expression induced by bilirubin. Bilirubin and LCA have damaging consequences in osteocytes by decreasing viability, differentiation and mineralization, increasing apoptosis and modifying gene expression, effects that are neutralized by UDCA.

PKC-\u03b4 deficiency in B cells displays osteopenia accompanied with upregulation of RANKL expression and osteoclast\u2013osteoblast uncoupling

PKC-δ is an important molecule for B-cell proliferation and tolerance. B cells have long been recognized to play a part in osteoimmunology and pathological bone loss. However, the role of B cells with PKC-δ deficiency in bone homeostasis and the underlying mechanisms are unknown. We generated mice with PKC-δ deletion selectively in B cells by crossing PKC-δ-loxP mice with CD19-Cre mice. We studied their bone phenotype using micro-CT and histology. Next, immune organs were obtained and analyzed. Western blotting was used to determine the RANKL/OPG ratio in vitro in B-cell cultures, ELISA assay and immunohistochemistry were used to analyze in vivo RANKL/OPG balance in serum and bone sections respectively. Finally, we utilized osteoclastogenesis to study osteoclast function via hydroxyapatite resorption assay, and isolated primary calvaria osteoblasts to investigate osteoblast proliferation and differentiation. We also investigated osteoclast and osteoblast biology in co-culture with B-cell supernatants. We found that mice with PKC-δ deficiency in B cells displayed an osteopenia phenotype in the trabecular and cortical compartment of long bones. In addition, PKC-δ deletion resulted in changes of trabecular bone structure in association with activation of osteoclast bone resorption and decrease in osteoblast parameters. As expected, inactivation of PKC-δ in B cells resulted in changes in spleen B-cell number, function, and distribution. Consistently, the RANKL/OPG ratio was elevated remarkably in B-cell culture, in the serum and in bone specimens after loss of PKC-δ in B cells. Finally, in vitro analysis revealed that PKC-δ ablation suppressed osteoclast differentiation and function but co-culture with B-cell supernatant reversed the suppression effect, as well as impaired osteoblast proliferation and function, indicative of osteoclast–osteoblast uncoupling. In conclusion, PKC-δ plays an important role in the interplay between B cells in the immune system and bone cells in the pathogenesis of bone lytic diseases.

Effect of interleukin-22 on osteogenic differentiation and the osteoclastogenic response of human periodontal ligament fibroblasts in vitro.

Interleukin-22 (IL-22) exerts extensive biological effects, playing both protective and pathological roles in autoimmune and infectious diseases. However, the specific role and mechanism of IL-22 in the pathogenesis of periodontitis have not been clarified. The aim of this study was to analyze the possible roles of IL-22 in the osteoclastogenesis and osteogenesis of periodontitis. Human periodontal ligament fibroblasts (hPDLFs) were treated with IL-22 and/or lipopolysaccharide from Porphyromonas gingivalis (Pg-LPS), and the mRNA and protein expression of RANKL and OPG were measured by qRT-PCR and Western blotting, respectively. Western blotting was also used to examine the phosphorylated and total protein expression of MAPK signaling molecules. The role of the MAPK pathway in osteoclastogenesis marker expression was further confirmed by inhibition assays. For osteogenic assays, the mRNA expression of osteoblastic markers was quantified by qRT-PCR, the alkaline phosphatase (ALP) activity of hPDLFs was measured by an ALP assay, and the mineralized nodules formed by hPDLFs were determined by Alizarin Red S staining. IL-22 promoted the expression of RANKL in hPDLFs via the MAPK signaling pathway and further upregulated RANKL expression together with Pg-LPS via the p38 MAPK pathway. IL-22 could enhance the ALP activity and mineralized nodule formation of hPDLFs in the early period of osteogenic induction, while exhibiting no profound effect on the expression of osteoblastic markers. IL-22 plays regulatory roles in bone homeostasis, and it is likely to contribute to osteoclastogenesis as a proinflammatory cytokine in the pathogenesis of periodontitis.

RANKL blockade suppresses pathological angiogenesis and vascular leakage in ischemic retinopathy

Receptor activator of NF-κB ligand (RANKL) is a member of the TNF superfamily. RANKL increases endothelial permeability and induces angiogenesis, suggesting its critical roles in the vasculature. Despite the evidence implicating RANKL in vascular pathology, its role in ischemic retinopathy has not been previously reported. In this study, neonatal mice were exposed to 75% oxygen from postnatal day (P)7 to P12 to induce vaso-obliteration, and then returned to room air from P12 to P17, causing the retina to become hypoxic and inducing vascular endothelial growth factor (VEGF) signaling, which produces pathological neovascularization. On P12, the mice received a single intravitreal injection of control IgG1 or RANK-Fc, and retinas were obtained at P17. On P17, RANKL was expressed strongly and selectively in the neovascular tufts (NVT) area. RANKL colocalized with αSMA or PDGFRβ in NVT. However, co-immunostaining revealed that CD31-positive areas were not the same as RANKL, which indicates that RANKL might be produced by retinal pericytes, not endothelial cells. Consistent with this finding, chemical hypoxia upregulated RANKL expression in cultured human retinal pericytes but not in endothelial cells. Treatment with RANK-Fc markedly reduced the NVT area compared to that in mice administered the IgG1 injection. In contrast, the central avascular region of RANKL-Fc retina was comparable to the controls. In addition, we assessed retinal vascular permeability using FITC-labeled dextran. RANK-Fc treated mice displayed decreased vascular leakages compared to those injected with IgG1. Our work supports the use of an RANKL blockade as a potential therapeutic approach against ischemic retinopathies.

Vibration enhances osteoclastogenesis by inducing RANKL expression via NF-κB signaling in osteocytes.

To shorten the duration of orthodontic treatment it is important not only to reduce risks such as dental caries, periodontal disease, and root resorption, but also to decrease pain and discomfort caused by a fixed appliance. Several studies have investigated the effect of vibration applied to fixed appliances to accelerate tooth movement. Although it was reported that vibration accelerates orthodontic tooth movement by enhancing alveolar bone resorption, the underlying cellular and molecular mechanisms remain unclear. In this study, we investigated the effects of vibration on osteoclastogenesis in vitro and in vivo. Vibration applied to pre-osteoclast cell line RAW264.7 cells enhanced cell proliferation but did not affect their differentiation into osteoclasts. Osteocytes in bone are known to be mechanosensitive and to act as receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL). Therefore, in the present study, vibration was applied to cells from the osteocyte-like cell line MLO-Y4. In MLO-Y4 cells, vibration induced phosphorylation of the inhibitor of NF-κB (IκB) and caused nuclear localization of NF-κB p65. Additionally, vibration increased RANKL mRNA expression, but did not affect osteoprotegerin (OPG) mRNA expression in MLO-Y4 cells, thus resulting in an increased RANKL/OPG ratio. Consistent with these findings, vibration applied during experimental tooth movement increased NF-κB activation and RANKL expression in osteocytes on the compression side of alveolar bone in vivo, whereas vibration had no such effects on the tension side. Furthermore, in a co-culture of MLO-Y4 cells and RAW264.7 cells, vibration applied to MLO-Y4 cells enhanced osteoclastogenesis. These findings suggest that vibration could accelerate orthodontic tooth movement by enhancing osteoclastogenesis through increasing the number of pre-osteoclasts and up-regulating RANKL expression in osteocytes on the compression side of alveolar bone via NF-κB activation.

Berberine Chloride and Hyperthermia Promote Osterix Expression and Suppress Cell Cycle Genes in Osteosarcoma Cells

Objectives: The aim of the current study was to investigate the effects of berberine chloride and various heat conditions on the gene expression of Osterix, RUNX-2, RANKL, CDK2, CDK4, IL-6 and IL-11.
 Methods: Six groups of cells were treated with hyperthermia for 1 h: Two groups at mild, two at moderate and two at severe hyperthermia (39, 43 and 45°C respectively). Berberine chloride (80 µg/mL) was selected for treating one group of mild, moderate and severe hyperthermia (combination). All treated groups were recovered at 37°C for 24 h. Another exposure for hyperthermia (1 h) and recovery for 3 h at 37°C were applied.
 Results: The expression of Osterix was highly upregulated in all groups except in the severe hyperthermia and mild hyperthermia with berberine groups. Only the mild hyperthermia without berberine induced a slight increase in the expression of RUNX2, whereas severe hyperthermia alone suppressed the levels on a significant manner. Berberine alone was more effective in significantly up-regulating RANKL expression. On the other hand, CDK2 mRNA was downregulated in all groups. CDK4 showed a similar regulation in the mild hyperthermia group with control, but the expression was downregulated in the other groups, especially in severe hyperthermia the expression was significantly downregulated (p<0.5). IL-6 was upregulated highly and significantly in the group of berberine and all groups of combinations, whereas mild and moderate hyperthermia stimulated significant expression of IL-11 mRNA.
 Conclusion: These results suggest that hyperthermia and berberine chloride can promote osteosarcoma cells differentiation and arrest cell-cycle.

Berberine Chloride and Hyperthermia Promote Osterix Expression and Suppress Cell Cycle Genes in Osteosarcoma Cells

Objectives: The aim of the current study was to investigate the effects of berberine chloride and various heat conditions on the gene expression of Osterix, RUNX-2, RANKL, CDK2, CDK4, IL-6 and IL-11.
 Methods: Six groups of cells were treated with hyperthermia for 1 h: Two groups at mild, two at moderate and two at severe hyperthermia (39, 43 and 45°C respectively). Berberine chloride (80 µg/mL) was selected for treating one group of mild, moderate and severe hyperthermia (combination). All treated groups were recovered at 37°C for 24 h. Another exposure for hyperthermia (1 h) and recovery for 3 h at 37°C were applied.
 Results: The expression of Osterix was highly upregulated in all groups except in the severe hyperthermia and mild hyperthermia with berberine groups. Only the mild hyperthermia without berberine induced a slight increase in the expression of RUNX2, whereas severe hyperthermia alone suppressed the levels on a significant manner. Berberine alone was more effective in significantly up-regulating RANKL expression. On the other hand, CDK2 mRNA was downregulated in all groups. CDK4 showed a similar regulation in the mild hyperthermia group with control, but the expression was downregulated in the other groups, especially in severe hyperthermia the expression was significantly downregulated (p<0.5). IL-6 was upregulated highly and significantly in the group of berberine and all groups of combinations, whereas mild and moderate hyperthermia stimulated significant expression of IL-11 mRNA.
 Conclusion: These results suggest that hyperthermia and berberine chloride can promote osteosarcoma cells differentiation and arrest cell-cycle.

Intraarticular injection of dexamethasone promotes bone erosion in collagen-induced arthritis in mice through up-regulation of RANKL expression.

Dexamethasone (DEX) is an effective therapeutic option commonly used in the treatment of many inflammatory diseases. However, DEX could impair proliferation or differentiation of osteoblasts, suggesting a pivotal role of DEX in bone destruction. To investigate whether intraarticular injection of DEX could exacerbate bone erosion during CIA development. Collagen-induced arthritis (CIA) mice were divided into PBS-treated and DEX-treated groups (n = 5/group). Negative control group: DBA/1 mice (n = 5) were used as age-matched, healthy, untreated controls. CIA was induced in male DBA/1 mice. Intraarticular injected DEX (0.01mg/Kg, 10μl) into the knee joint of CIA on Day 28, Day 35, Day 42 and Day 49 post the 1st immunization. The severity of the arthritic disease was ameliorated in DEX-treated mice, accompanied by the decreased expression of IL-6, IL-8 and TNF-α. However, DEX treatment accelerates bone erosion and osteoporosis during CIA development and triggers higher expression of RANKL, IL-17 in vitro and vivo. The effect of DEX on bone structure was analyzed using Haematoxylin & Eosin (H&E) staining and Micro-CT. The levels of receptor activator for nuclear factor-κ B ligand (RANKL) and osteoprotegerin (OPG) were investigated by real-time PCR, Western Blot and immunohistochemical analysis. RASFs were stimulated with Interleukin (IL)-1β and then treated with different concentrations of DEX for 72h. Intraarticular injection of DEX could exacerbate bone erosion in CIA model via up-regulation of RANKL expression.

Magnolol Inhibits Osteoclast Differentiation via Suppression of RANKL Expression

Magnolol, a compound from the traditional Korean herb Magnolia sp., has been exhaustively investigated as a therapeutic agent against several diseases including systemic and local inflammation. We examined the effects of magnolol on osteoclastic differentiation associated with inflammation. Magnolol markedly reduced interleukin (IL)-1-induced osteoclast formation in co-cultures of murine osteoblasts and bone marrow cells, whereas it had no effect on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation in bone marrow macrophage cultures. In osteoblasts, magnolol markedly inhibited both the up-regulation of RANKL expression and the production of prostaglandin E2 (PGE2) in response to IL-1 treatment. Addition of exogenous PGE2 reversed the inhibitory effects of magnolol on IL-1-induced RANKL expression in osteoblasts and osteoclast formation in co-cultures. Magnolol inhibited IL-1-induced PGE2 production, at least in part by suppressing cyclooxygenase-2 (COX-2) expression. Taken together, these results demonstrate that magnolol inhibits IL-1-induced RANKL expression in osteoblasts through suppression of COX-2 expression and PGE2 production, resulting in inhibition of osteoclast differentiation in co-cultures.

Effect of high-frequency near-infrared diode laser irradiation on periodontal tissues during experimental tooth movement in rats.

Tooth movement during orthodontic treatment is associated with bone neoplasticity and bone resorption on the tension and pressure sides. Previous clinical studies have suggested that low-power laser irradiation can accelerate tooth movement during orthodontic treatment, although the underlying mechanism remains unclear. In this study, we used a high-frequency near-infrared diode laser that generates less heat and examined the histologic changes in periodontal tissue during experimental tooth movement with laser irradiation. A nickel-titanium closed coil was mounted between the maxillary left side first molar and incisor of rats to model experimental tooth movement. The laser-irradiation and the control groups were set, and the amount of movement of the first molar on 7th and 14th days after the start of pulling of the first molar tooth on the maxillary left was measured by three-dimensional analysis of µCT. After tooth movement, tissue samples from the mesial and tension sides were collected, and successive horizontal sections were prepared and examined using hematoxylin-eosin and TRAP staining and immunohistochemical staining for RANKL, OPG, ALP, and proliferating cell nuclear antigen (PCNA). Changes in tissue temperature following laser irradiation were also examined. Laser irradiation significantly increased tooth movement compared with non-irradiated controls. Histologic staining of the pressure-side mesial root in laser-irradiated rats revealed enhanced RANKL expression and increased numbers of TRAP-positive cells compared with controls. By contrast, on the tension side, laser irradiation led to increased expression of ALP and PCNA. These data indicate that high-frequency near-infrared diode laser irradiation on the pressure side upregulates RANKL expression and accelerates osteoclast differentiation, facilitating bone resorption, whereas bone formation is induced on the tension side. This study demonstrates that high-frequency near-infrared diode laser irradiation of periodontal tissue leads to metabolic activation, which ultimately increases the rate of tooth movement. Lasers Surg. Med. © 2018 Wiley Periodicals, Inc.

Roles of VEGF-Flt-1 signaling in malignant behaviors of oral squamous cell carcinoma.

BackgroundVascular endothelial growth factor (VEGF) is a highly specific signaling protein for vascular endothelial cells that plays a critical role in tumor growth and invasion through angiogenesis, and may contribute to cell migration and activation of pre-osteoclasts, osteoclasts and some tumor cells.ObjectivesWe aimed to clarify the detailed roles of VEGF-Flt-1 signaling in bone invasion of oral squamous cell carcinoma (OSCC) cells.ResultsForty-two (42) of 54 cases with gingival SCC (77.8%) strongly expressed VEGF, and had a significantly increased number of Flt-1+ osteoclasts (p<0.01) and more aggressive bone invasion (p<0.05). PlGF, a ligand of Flt-1, induced osteoclastogenesis in single culture of bone marrow cells (BMCs), and inhibition of Flt-1-signaling by VEGF tyrosine kinase inhibitor and It’s down stream (Akt and ERK1/2) inhibitos reduced osteoclastogenesis in PlGF-stimulated BMCs (p<0.01). In molecular level, PlGF stimulation significantly upregulated RANKL expression in Flt-1-expressing HSC2 cells via phosphorylation of Akt and ERK1/2. In the co-culture of VEGF-producing HSC2 cells and BMCs, number of TRAP-positive osteoclasts markedly increased (p<0.01). The osteoclastogenesis was significantly inhibited by RANKL-neutralizing antibody (p<0.01) as well as by VEGF tyrosine kinase inhibitor (p<0.01) and it’s downstream (Akt and ERK1/2) inhibitors (p<0.01, p<0.05, respectively).ConclusionVEGF-Flt-1 signaling induces osteoclastogenesis in OSCC through two possible ways: 1) VEGF produced from OSCC cells can directly stimulate the Flt-1 pathway in preosteoclasts to induce migration to future bone resorbing area and differentiation into osteoclasts, and 2) VEGF-Flt-1 signaling upregulates RANKL expression in OSCC cells, which indirectly leads to osteoclast differentiation. Therefore, blocking of the VEGF-Flt-1 signaling may help inhibit bone invasion of OSCC.

Interleukin 17 regulates SHP-2 and IL-17RA/STAT-3 dependent Cyr61, IL-23 and GM-CSF expression and RANKL mediated osteoclastogenesis by fibroblast-like synoviocytes in rheumatoid arthritis

Interleukin (IL)-17 predominately produced by the Th17 cells, plays a crucial role in the fibroblast-like synoviocytes (FLS) mediated disease process of rheumatoid arthritis (RA). IL-17 exerts its pathogenic effects in RA-FLS by IL-17/IL-17RA/STAT-3 signaling. Recent studies have shown that RA-FLS produces SHP-2, Cyr61, IL-23, GM-CSF and RANKL which results in worsening of the disease. However, whether IL-17/IL-17RA/STAT-3 signaling regulates SHP-2, Cyr61, IL-23, GM-CSF and RANKL expressions in RA-FLS remains unknown. In this study, IL-17 treatment dramatically induced the production of Cyr61, IL-23 and GM-CSF in FLS isolated from adjuvant induced arthritis (AA) rats. Conversely, IL-17 mediated production of Cyr61, IL-23 and GM-CSF was abrogated by knockdown of IL-17RA using a small interfering RNA or blockade of STAT-3 activation with S3I-201 in AA-FLS. Interestingly, IL-17 treatment noticeably increased the expression of IL-17RA and SHP-2 in AA-FLS. However, silencing of IL-17RA reversed the effect of IL-17 on the expression of IL-17RA and SHP-2 in AA-FLS. In addition, an increased number of TRAP-positive multinucleated cells were observed in a coculture system consisting of IL-17 treated AA-FLS and rat bone marrow derived monocytes/macrophages. Further, mechanistically we found that IL-17 upregulated RANKL expression in AA-FLS that was dependent on the IL-17/IL-17RA/STAT-3 signaling cascade. Knockdown of IL-17RA or inhibition of STAT-3 activation decreased the IL- 17 induced RANKL expression by AA-FLS and their osteoclastogenic potential. Taken together, our findings demonstrate that IL-17 regulates SHP-2 expression and IL-17RA/STAT-3 dependent production of Cyr61, IL-23, GM-CSF and RANKL in AA-FLS and may reveal a new insight into the pathogenesis of RA.

Lipopolysaccharide increases IL-6 secretion via activation of the ERK1/2 signaling pathway to up-regulate RANKL gene expression in MLO-Y4 cells.

Lipopolysaccharide (LPS) plays an important role in bone resorption, which involves numerous cytokines through various signaling pathways. RANKL and interleukin (IL)-6 are two important cytokines that are involved in bone remodeling. The aim of this study was to evaluate the effect of LPS on RANKL and IL-6 gene expression, the relationship of RANKL and IL-6, and the role of extracellular signal-regulated kinases 1/2 (ERK1/2) on IL-6 secretion induced by LPS in MLO-Y4 cells. The cells were stimulated by LPS at different concentrations (1, 10, 100, 500, and 1000 ng/mL) for different durations (0.5, 1, 2, 4, and 8 h and 0.5, 1, 1.5, 2, and 4 h), and the mRNA expressions of RANKL and IL-6 were determined by PCR. In the presence of 100 ng/mL LPS at different time points (0.5, 1, 1.5, 2, and 4 h), IL-6 secretion and ERK1/2 phosphorylation in the cells were determined by ELISA and western blotting, respectively. STAT3 phosphorylation in cells simulated by 100 ng/mL LPS at different time points (0.5, 1, 2, 4, and 8 h) was assessed by western blotting. We found that LPS significantly up-regulated RANKL expression and activated the ERK1/2 pathway to induce IL-6 mRNA expression and protein synthesis in MLO-Y4 cells. However, the increased IL-6 was blocked by pre-treatment of MLO-Y4 cells with the ERK1/2 inhibitor U0126 (10 µM), and the enhanced RANKL was blocked by the STAT3 inhibitor S3I-201 (100 µM). Our results indicate that LPS up-regulates osteocyte expression of RANKL and IL-6, and the increased RANKL is associated with the up-regulation of IL-6, which involves the ERK1/2 pathway.