Receptor Activator Of NF-kappaB Ligand mRNA Expression Research Articles

Wnt5a-Ror2 signaling mediates root resorption

This study aimed to investigate the role of wingless-type MMTV integration site family member 5a (Wnt5a)-receptor tyrosine kinase-like orphan receptor 2 (Ror2) signaling in root resorption. The messenger RNA (mRNA) expression of Wnt5a, Ror2, and RANKL in periodontal ligament cells (PDLCs) under compression force (CF) with or without Ror2 small interfering RNA (siRNA) were measured by quantitative reverse transcription-polymerase chain reaction, and these proteins released into culture supernatants were measured using enzyme-linked immunosorbent assay. Then these PDLC-conditioned media under CF with or without Ror2 siRNA were used to culture osteoclast precursors to detect osteoclastogenesis effects via tartrate-resistant acid phosphatase staining. In invivo studies, the odontoclast number and the root resorption volume under excessive CF with or without Ror2 siRNA were investigated by tartrate-resistant acid phosphatase immunohistochemical staining and microcomputed tomography. The protein levels for Wnt5a, Ror2, and receptor activator of nuclear factor-kappa B ligand (RANKL) in the periodontal ligament tissues were also detected using immunohistochemical staining. Finally, the odontoclast number, root resorption volume, and the mRNA and protein expressions were compared between immature and mature teeth. The mRNA production and protein release level of Wnt5a, Ror2, and RANKL increased after CF, whereas they were significantly downregulated with Ror2 siRNA. The osteoclast number increased treating with culture medium from PDLC applying CF, but the increase was inhibited after adding Ror2 siRNA. In the animal model, the odontoclast number and root resorption volume significantly increased in the CF group but decreased in the CF with the Ror2 siRNA group. The protein levels of Wnt5a, Ror2, and RANKL in periodontal ligament were upregulated under excessive CF, and the pathway was inhibited with Ror2 siRNA. In the immature tooth group, the odontoclast number, root resorption volume, and the mRNA and protein expressions of Wnt5a-Ror2 signaling were reduced. Wnt5a-Ror2 signaling in PDLCs enhanced by excessive CF could promote RANKL release and induce precursor differentiation, partly leading to increased odontoclast activity and ultimate root resorption. The less resorption of the immature tooth may be due to odontoclastogenesis inhibition by decreased expression of Wnt5a-Ror2 signaling.

Preliminary investigation on the molecular mechanisms underlying the correlation between VDR-FokI genotype and periodontitis.

The only polymorphism that could change the protein structure in vitamin D receptor (VDR) is the FokI polymorphism (rs2228570). The FF genotype has the strongest transcriptional activity of VDR and is correlated with higher susceptibility to periodontitis. To reveal the possible molecular mechanisms for the correlation preliminarily, the influence of VDR-FokI genotype on the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) in human gingival fibroblasts (hGFs) and human periodontal ligament cells (hPDLCs) was investigated in this study. hGFs and hPDLCs from 15 donors (five FF, seven Ff, and three ff) were treated with 1,25OH2 D3 , with or without the specific knockdown of VDR using siRNA. The mRNA and protein expression of OPG and RANKL were detected using real-time PCR and enzyme-linked immunosorbent assay, respectively. Both in hGFs and hPDLCs, 1,25OH2 D3 could significantly induce the mRNA and protein expression of RANKL, and FF genotype had significantly higher induction than the other genotypes, however, neither 1,25OH2 D3 nor VDR-FokI had significant influence on the OPG expression. As a result, the RANKL/OPG ratio was significantly elevated under 1,25OH2 D3 stimulation and FF genotype had the most remarkable elevation. When VDR was knocked down, all the differences among the three genotypes disappeared. The strongest transcriptional activity of FF genotype might contribute to the strongest enhancement of RANKL expression and RANKL/OPG ratio in hGFs and hPDLCs stimulated by 1,25OH2 D3 , which might help to reveal the mechanisms of the correlation between FF genotype and susceptibility to periodontitis.

Abstract 1970: Melatonin inhibits osteolytic bone metastasis through interrupting cancer cells-derived osteolytic factor and reduce osteoclast differentiation

Abstract Osteolytic bone metastasis is seen in lung, breast, and prostate cancer and is associated with severe complications such as bone pain, fractures and hypercalcemia. In the bone microenvironment, osteoclasts play an important role in bone remodeling and bone resorption. Metastatic cancer cells may increase osteoclast differentiation and activity by releasing inflammatory cytokines including receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and IL-8, leading to bone resorption and osteolytic lesions. The endogenous hormone melatonin is known to modulate bone resorption. Increasing evidence suggests that melatonin inhibits cancer cell proliferation, angiogenesis and metastasis. Up until now, the inhibitory role of melatonin in osteolytic bone metastasis has remained unclear. In In vitro study, we observed that melatonin inhibits the differentiation of precursor osteoclasts and promotes apoptosis in mature osteoclasts. We injected mice tibia with luciferase-labeled osteolytic cancer cell lines (lung cancer: A549, prostate cancer: PC-3) to establish bone metastasis. After 4 weeks of intraperitoneal injection with melatonin, IVIS fluorescence signaling indicated that melatonin inhibited tumor growth. X-ray imaging of the bone revealed an improvement in bone resorption after melatonin treatment. In In vitro melatonin also reduced RANKL protein and mRNA expression in the A549 and PC-3 cell lines. Our data indicate that melatonin improves bone mass by inhibiting the expression of cancer-derived osteolytic factors and by promoting apoptosis in mature osteoclasts. Our future investigations will seek to determine the signaling pathway involved in melatonin-regulated RANKL expression in cancer cells. In summary, our findings provide insight into the benefits of melatonin treatment in osteolytic bone metastasis. Citation Format: Jyun-Lin Lai, An-Chen Chang, Po-Chun Chen, Chih-Hsin Tang. Melatonin inhibits osteolytic bone metastasis through interrupting cancer cells-derived osteolytic factor and reduce osteoclast differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1970.

Effect of TNF-α-Induced Sclerostin on Osteocytes during Orthodontic Tooth Movement.

Osteocytes are abundant cells in bone, which contribute to bone maintenance. Osteocytes express receptor activator of nuclear factor kappa-B ligand (RANKL) and regulate osteoclast formation. Orthodontic tooth movement (OTM) occurs by osteoclast resorption of alveolar bone. Osteocyte-derived RANKL is critical in bone resorption during OTM. Additionally, tumor necrosis factor-α (TNF-α) is important in osteoclastogenesis during OTM. Sclerostin has been reported to enhance RANKL expression in the MLO-Y4 osteocyte-like cell line. This study investigated the effect of TNF-α on sclerostin expression in osteocytes during OTM. In vitro analysis of primary osteocytes, which were isolated from DMP1-Topaz mice by sorting the Topaz variant of GFP-positive cells, revealed that SOST mRNA expression was increased when osteocytes were cultured with TNF-α and that RANKL mRNA expression was increased when osteocytes were cultured with sclerostin. Moreover, the number of TRAP-positive cells was increased in osteocytes and osteoclast precursors cocultured with sclerostin. In vivo analysis of mouse calvariae that had been subcutaneously injected with phosphate-buffered saline (PBS) or TNF-α revealed that the number of TRAP-positive cells and the percentage of sclerostin-positive osteocytes were higher in the TNF-α group than in the PBS group. Furthermore, the level of SOST mRNA was increased by TNF-α. As an OTM model, a Ni-Ti closed-coil spring connecting the upper incisors and upper-left first molar was placed to move the first molar to the mesial direction in wild-type (WT) mice and TNF receptor 1- and 2-deficient (TNFRsKO) mice. After 6 days of OTM, the percentage of sclerostin-positive osteocytes on the compression side of the first molar in TNFRsKO mice was lower than that in WT mice. In this study, TNF-α increased sclerostin expression in osteocytes, and sclerostin enhanced RANKL expression in osteocytes. Thus, TNF-α may play an important role in sclerostin expression in osteocytes and enhance osteoclast formation during OTM.

Effect of esculetin on bone metabolism in ovariectomized rats

OBJECTIVETo determine the effect of an esculetin formulation (at 97.4% purity) on osteoporosis, and to investigate the potential underlying molecular mechanism(s). METHODSSixty specific pathogen free-grade female Wistar rats were randomly assigned to three groups: blank control (n = 12), sham (n = 12), and model (n = 36). The model group were bilaterally ovariectomized. The sham group had the tissue surrounding the ovaries removed, while the ovaries were retained. After 3 months, the model group was randomly divided into three subgroups: OVX (n = 12), positive control (n = 12), and esculetin (n = 12). The positive control group and the esculetin group were intragastrically administered diethylstilbestrol (0.046 mg • kg−1 • d−1) or esculetin (384 mg • kg−1 • d−1), respectively, once per day for 6 consecutive days; medication administration was then stopped for 1 d, before being administered for another 6 consecutive days. All rats were treated for 3 months. Samples were collected at the end of the treatment period. An Osteocore3 Digital 2D bone densitometer was used to test the bone mineral density, and histomorphometric analysis was performed to measure bone mass, bone formation, and bone resorption. Enzyme-linked immunosorbent assay analysis was used to measure the serum concentrations of interleukin-6 (IL-6), osteoprotegerin (OPG), and receptor activator of nuclear factor-kappa B ligand (RANKL). Immunohistochemistry and in situ hybridization were performed to detect the protein and mRNA expressions of OPG and RANKL in osteoblasts and bone marrow stromal cells. RESULTSCompared with the OVX group, the esculetin group had significantly greater femoral bone mineral density and tibial trabecular bone volume, and significantly smaller trabecular resorption surface. The percentage of trabecular formation surface, average osteoid width, trabecular bone mineralization rate, and cortical bone mineralization rate did not significantly differ between groups. Compared with the sham group, the esculetin group had significantly decreased serum levels of IL-6 and RANKL, and significant downregulation of RANKL protein and mRNA expression levels in osteoblasts and bone marrow stromal cells; however, there was no significant difference between groups in OPG. CONCLUSIONEsculetin can increase bone mass by upregulating RANKL expression in osteoblasts and bone marrow stromal cells, and decreasing serum IL-6 concentration. This indicates that the therapeutic effect of esculetin on osteoporosis occurs via decreased bone resorption.

TRPM3/TRPV4 regulates Ca2+-mediated RANKL/NFATc1 expression in osteoblasts

Mechanical stress plays an important role in the regulation of bone turnover. However, the mechanism underlying hypo-osmotic stress-induced cellular response in osteoblasts remains poorly understood. In this study, we investigated the effect of hypotonic stress on the expression of bone remodeling factors, including the receptor activator of nuclear factor-kappa B ligand (RANKL) and the nuclear factor of activated T cells type c1 (NFATc1) in primary mouse osteoblasts and MC3T3-E1 cells. Hypo-osmotic stress induced significant increases in RANKL mRNA expression and intracellular Ca2+ concentration ([Ca2+]i) from the extracellular space. Hypo-osmotic stress-induced effects on [Ca2+]i and RANKL and NFATc1 protein expression were decreased by antagonists of transient receptor potential melastatin 3 (TRPM3) and vanilloid 4 (TRPV4). Agonists of TRPM3 and TRPV4 activated [Ca2+]i and RANKL and NFATc1 protein expression. Furthermore, genetic suppression of Trpm3 and Trpv4 reduced hypo-osmotic stress-induced effects in mouse osteoblasts. These results suggest that hypo-osmotic stress induces increases in [Ca2+]i through TRPM3 and TRPV4 to regulate RANKL and NFATc1 expression in mouse osteoblastic cells and that mechanical stress-activated TRP channels may play a critical role in bone remodeling.

P38 MAPK and Its Upstream Activators MKK3 and MKK6 Play an Important Role on Lipopolysaccharide-Induced RANKL Expression by Periodontal Ligament Cells

Resident, non-immune cells express various pattern-recognition receptors and produce inflammatory cytokines in response to microbial antigens, during the innate immune response. Alveolar bone resorption is the hallmark of destructive periodontitis and it is caused by the host response to bacteria and their mediators present on the biofilm. The balance between the expression levels of receptor activator of nuclear factorkappa B ligand (RANKL) and osteoprotegerin (OPG) is pivotal for osteoclast differentiation and activity and has been implicated in the progression of bone loss in periodontitis. To assess the contribution of resident cells to the bone resorption mediated by innate immune signaling, we stimulated fibroblasts and osteoblastic cells with LPS from. Escherichia coli (TLR4 agonist), Porphyromonas gingivalis (TLR2 and -4 agonist), and interleukin-1 beta (as a control for cytokine signaling through Toll/IL-1receptor domain) in time-response experiments. Expression of RANKL and OPG mRNA was studied by RT-PCR, whereas the production of RANKL protein and the activation of p38 MAPK and NF-kB signaling pathways were analyzed by western blot. We used biochemical inhibitors to assess the relative contribution of p38 MAPK and NF-kB signaling to the expression of RANKL and OPG induced by TLR2, -4 and IL1β in these cells. Both p38 MAPK and NFkB pathways were activated by these stimuli in fibroblasts and osteoblasts, but the kinetics of this activation varied in each cell type and with the nature of the stimulation. E. coli LPS was a stronger inducer of RANKL mRNA in fibroblasts, whereas LPS from P. gingivalis downregulated RANKL mRNA in periodontal ligament cells but increased its expression in osteoblasts. IL-1β induced RANKL in both cell types and without a marked effect on OPG expression. p38 MAPK was more relevant than NF-kB for the expression of RANKL and OPG in these cell types.

Ameloblastin and enamelin prevent osteoclast formation by suppressing RANKL expression via MAPK signaling pathway

Ameloblastin (Ambn) and enamelin (Enam) play a pivotal role in enamel mineralization. Previous studies have demonstrated that these enamel-related gene products also affect bone growth and remodeling; however, the underlying mechanisms have not been elucidated. In the present study, we examined the effects of Ambn and Enam on the receptor activator of nuclear factor kappa-B ligand (RANKL) expression induced with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and dexamethasone (DEX) on mouse bone marrow stromal cell line ST2 cells. We then verified the effect of Ambn and Enam on osteoclastogenesis. We found that pretreatment with recombinant human Ambn (rhAmbn) and recombinant human Enam (rhEnam) remarkably suppressed RANKL mRNA and protein expression induced with 1,25(OH)2D3 and DEX. Interestingly, rhAmbn and rhEnam attenuated the phosphorylation of mitogen-activated protein kinases (MAPK), including ERK1/2, JNK, and p38 in ST2 cells stimulated with 1,25(OH)2D3 and DEX. Moreover, pretreatment with specific inhibitors of ERK1/2 and p38, but not JNK, blocked RANKL mRNA and protein expression. Cell co-culture results showed that rhAmbn and rhEnam downregulated mouse bone marrow cell differentiation into osteoclasts induced with 1,25(OH)2D3 and DEX-stimulated ST2 cells. These results suggest that Ambn and Enam may indirectly suppress RANKL-induced osteoclastogenesis via downregulation of p38 and ERK1/2 MAPK signaling pathways in bone marrow stromal cells.

Drug-releasing nano-engineered titanium implants: therapeutic efficacy in 3D cell culture model, controlled release and stability

There is an ongoing demand for new approaches for treating localized bone pathologies. Here we propose a new strategy for treatment of such conditions, via local delivery of hormones/drugs to the trauma site using drug releasing nano-engineered implants. The proposed implants were prepared in the form of small Ti wires/needles with a nano-engineered oxide layer composed of array of titania nanotubes (TNTs). TNTs implants were inserted into a 3D collagen gel matrix containing human osteoblast-like, and the results confirmed cell migration onto the implants and their attachment and spread. To investigate therapeutic efficacy, TNTs/Ti wires loaded with parathyroid hormone (PTH), an approved anabolic therapeutic for the treatment of severe bone fractures, were inserted into 3D gels containing osteoblast-like cells. Gene expression studies revealed a suppression of SOST (sclerostin) and an increase in RANKL (receptor activator of nuclear factor kappa-B ligand) mRNA expression, confirming the release of PTH from TNTs at concentrations sufficient to alter cell function. The performance of the TNTs wire implants using an example of a drug needed at relatively higher concentrations, the anti-inflammatory drug indomethacin, is also demonstrated. Finally, the mechanical stability of the prepared implants was tested by their insertion into bovine trabecular bone cores ex vivo followed by retrieval, which confirmed the robustness of the TNT structures. This study provides proof of principle for the suitability of the TNT/Ti wire implants for localized bone therapy, which can be customized to cater for specific therapeutic requirements.

Immune-Inflammatory Cell Profile and Receptor Activator of Nuclear Factor Kappa B Ligand/Osteoprotegerin Expression in Persistent Apical Periodontitis after Root Canal Retreatment Failure

IntroductionThis study assessed the immune-inflammatory profile and the expression of bone resorption activators receptor activator of nuclear factor kappa B ligand (RANKL) and inhibitor osteoprotegerin (OPG) in apical periodontitis (n = 20) that persisted after root canal retreatment. MethodsImmunohistochemistry was used to characterize lymphocyte populations (CD3+, CD45RO+, CD8+, and FoxP3+ cells), macrophages (CD68+), RANKL+ and OPG+ cells in persistent apical periodontitis (PAP) and primary periapical lesions (PPLs). By using quantitative real-time polymerase chain reaction, the mRNA expression of RANKL and OPG in PAP and periodontal ligament from healthy teeth was comparatively analyzed. The data were analyzed by Mann-Whitney, Pearson χ2, and Wilcoxon tests (5% level). ResultsPAP showed an elevated number of FoxP3+ cells compared with PPL (P < .001). The number of CD68+ cells was reduced in the PAP samples compared with the PPLs (P < .001). Similar number of other lymphocyte populations was observed in PAP and PPLs (P > .05 for all comparisons). No differences in the RANKL, OPG, and immune-inflammatory cells were demonstrated when comparing PAP microscopically classified as cyst with those classified as granulomas (P > .05 for all comparisons). The assessment of mRNA expression revealed higher levels of RANKL and OPG in PAP compared with the periodontal ligament from healthy teeth (control) samples (P < .001). Also, a greater expression of RANKL in comparison with OPG was observed in PAP (P < .001). ConclusionsThese findings indicate that PAP consists of biologically active lesions that demonstrate potential of bone resorption (higher expression of RANKL) and is characterized by an immune-inflammatory cell profile that suggests a suppressive and regulatory environment (higher number of FoxP3+ cells and lower number of macrophages) favorable to more chronic clinical behavior.

Attenuation of RANKL-induced Osteoclast Formation via p38-mediated NFATc1 Signaling Pathways by Extract ofEuphorbia LathyrisL

BackgroundOsteoclasts are the only cell type capable of breaking down bone matrix, and its excessive activation is responsible for the development of bone-destructive diseases. Euphorbia lathyris L. (ELL) is an herbal plant that belongs to the Euphorbiaceae family. This study investigated the effects of the methanol extract of the aerial part of ELL on receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast formation and signaling pathways.MethodsOsteoclasts were formed by co-culturing mouse bone marrow with osteoblasts or by culturing mouse bone marrow-derived macrophages (BMMs) with macrophage colony-stimulating factor (M-CSF) and RANKL. Bone resorption assays were performed using dentine slices. The expression level of mRNA was analyzed by real-time polymerase chain reaction (PCR) or reverse transcription (RT)-PCR. Western blotting assays were performed to detect the expression or activation level of proteins.ResultsELL inhibited RANKL-induced osteoclast formation without cytotoxicity. Furthermore, the RANKL-stimulated bone resorption was diminished by ELL. Mechanistically, ELL blocked the RANKL-triggered p38 mitogen-activated protein kinase (MAPK) phosphorylation, which resulted in the suppression of the expression of c-Fos and nuclear factor of activated T cells (NFATc1). In osteoblasts, ELL had little effect on the mRNA expression of RANKL and osteoprotegerin (OPG).ConclusionsThe present data suggest that ELL has an inhibitory effect on osteoclast differentiation and function via downregulation of the p38/c-Fos/NFATc1 signaling pathways. Thus, ELL could be useful for the treatment of bone diseases associated with excessive bone resorption.

Expression of neuropeptides and bone remodeling-related factors during periodontal tissue regeneration in denervated rats.

Increasing evidence indicates that bone remodeling is under the control of factors related to neuronal regulation, but the underlying mechanisms remain largely unknown. The aim of this study is attempted to assess the effect of denervation on neuropeptide expression and bone remodeling-related factors in the periodontal tissue regeneration process. rats underwent transection of the left inferior alveolar nerve (IAN-T) and surgery to produce bilateral periodontal defects; then, alveolar tissue was obtained from the animals of each group at 1, 2, 4, 6 and 8 weeks after the operation. The expression levels of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) were quantified by real-time PCR and Western blot techniques, and immunohistochemical staining was applied to investigate the in situ expression of OPG and RANKL. Osteoclasts were identified by tartrate-resistant acid phosphatase staining. The expression levels of calcitonin gene-related peptide (CGRP) mRNA and substance P (SP) mRNA were also quantified by real-time PCR. Mandibles with only periodontal defects were used as controls. After denervation, the OPG/RANKL ratio was reduced in the IAN-T groups due to decreased OPG mRNA and protein (P < 0.05) and a simultaneous increase in RANKL mRNA and protein expression (P < 0.05). On the other hand, the number of osteoclasts in periodontal defect areas increased, especially at 2 weeks, and at this time point, the expression of RANKL mRNA and protein peaked. The mRNA expression levels of neuropeptides CGRP and SP mRNA were monitored throughout the entire progress and exhibited a trend that was similar to that of the OPG/RANKL ratio, i.e., a downward trend over 1-2 weeks, an increase until 6 weeks, then back to the normal levels at 8 weeks. Innervation influences the OPG/RANKL ratio and neuropeptide expression, both of which govern the periodontal alveolar bone regeneration processes.

CCAAT/enhancer-binding protein β promotes receptor activator of nuclear factor-kappa-B ligand (RANKL) expression and osteoclast formation in the synovium in rheumatoid arthritis

IntroductionCCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor that is activated in the synovium in rheumatoid arthritis (RA) and promotes expression of various matrix metalloproteinases. In this study, we examined whether C/EBPβ mediates the expression of receptor activator of nuclear factor-kappa-B ligand (RANKL) and drives osteoclast formation in primary fibroblast-like synoviocytes (FLS) from RA patients. The cooperation of C/EBPβ and activation transcription factor-4 (ATF4) in the regulation of the RANKL promoter was also investigated.MethodsImmunofluorescence staining was performed for C/EBPβ, RANKL, and ATF4 in synovium from RA patients. Adenovirus expression vectors for two major isoforms, C/EBPβ-liver-enriched activator protein (LAP) and - liver-enriched inhibitory protein (LIP), or small interfering RNA for C/EBPβ, were used to manipulate C/EBPβ expression in RA-FLS. RA-FLS over-expressing C/EBPβ were co-cultured with peripheral blood mononuclear cells (PBMCs) to test osteoclast formation by tartrate-resistant acid phosphatase (TRAP) staining. A promoter assay for RANKL, a chromatin immunoprecipitation (ChIP) assay and an immunoprecipitation (IP) assay were also performed.ResultsImmunofluorescence staining showed colocalization of C/EBPβ, ATF4 and RANKL in RA synovium. Western blotting revealed the expression of C/EBPβ-LAP and -LIP in RA-FLS. Over-expression of either C/EBPβ-LAP or -LIP significantly increased the expression of RANKL mRNA, while C/EBPβ-LIP down-regulated osteoprotegerin (OPG) mRNA. The RANKL/OPG mRNA ratio was significantly increased by C/EBPβ-LIP over-expression. Knockdown of C/EBPβ with siRNA decreased the expression of RANKL mRNA. The number of TRAP-positive multinucleated cells was increased in co-cultures of PBMCs and FLS over-expressing either C/EBPβ-LAP or -LIP, but was more significant with LIP. C/EBPβ-LIP does not have a transactivation domain. However, promoter assays showed that C/EBPβ-LIP and ATF4 synergistically transactivate the RANKL promoter. ChIP and IP assays revealed the cooperative binding of C/EBPβ and ATF4 on the RANKL promoter.ConclusionsWe demonstrated that C/EBPβ, especially C/EBPβ-LIP in cooperation with ATF4, is involved in osteoclast formation by regulating RANKL expression in RA-FLS. These findings suggest that C/EBPβ plays a crucial role in bone destruction in RA joints.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0532-6) contains supplementary material, which is available to authorized users.

STAT-6 mediates TRAIL induced RANK ligand expression in stromal/preosteoblast cells

Receptor activator of nuclear factor kappa-B ligand (RANKL) is a critical osteoclastogenic factor expressed in bone marrow stromal/osteoblast lineage cells. Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) levels are elevated in pathologic conditions such as multiple myeloma and inflammatory arthritis, and have been positively correlated with osteolytic markers. Osteoprotegerin (OPG) which inhibits osteoclastogenesis is a decoy receptor for RANKL and also known to interact with TRAIL. Herein, we show that TRAIL increases DR5 and DcR1 receptors but no change in the levels of DR4 and DcR2 expression in human bone marrow derived stromal/preosteoblast (SAKA-T) cell line. We further demonstrated that TRAIL treatment significantly decreased OPG mRNA expression. Interestingly, TRAIL treatment induced RANKL mRNA expression in these cells. In addition, TRAIL significantly increased NF-kB and c-Jun N-terminal kinase (JNK) activity. Human transcription factor array screening by real-time RT-PCR identified TRAIL up-regulation of the signal transducers and activators of the transcription (STAT)-6 expression in SAKA-T cells. TRAIL stimulation induced p-STAT-6 expression in human bone marrow derived primary stromal/preosteoblast cells. Confocal microscopy analysis further revealed p-STAT-6 nuclear localization in SAKA-T cells. Chromatin immunoprecipitation (ChIP) assay confirmed p-STAT-6 binding to the hRANKL gene distal promoter region. In addition, siRNA suppression of STAT-6 expression inhibits TRAIL increased hRANKL gene promoter activity. Thus, our results suggest that TRAIL induces RANKL expression through a STAT-6 dependent transcriptional regulatory mechanism in bone marrow stromal/preosteoblast cells.

Effects of icariin on the regulation of the OPG-RANKL-RANK system are mediated through the MAPK pathways in IL-1β-stimulated human SW1353 chondrosarcoma cells.

Arthrodial cartilage degradation and subchondral bone remodeling comprise the most predominant pathological changes in osteoarthritis (OA). Moreover, accumulating evidence indicates that the abnormal expression of osteoprotegerin (OPG), receptor activator of nuclear factor kappa-B ligand (RANKL) and receptor activator of nuclear factor kappa-B (RANK) plays a vital role in the collapse of cartilage and subchondral bone. In the present study, the effects of icariin on the expression levels of these 3 factors in interleukin (IL)-1β-stimulated SW1353 chondrosarcoma cells were investigated. The SW1353 chondrosarcoma cells were cultured in the presence or absence of icariin and mitogen-activated protein kinase signaling pathway inhibitors, and were then stimulated with IL-1β. Cell viability was assessed by MTT assay. The mRNA and protein expression of OPG, RANKL and RANK was analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and ELISA, respectively. In addition, the levels of phosphorylated p38 (p-p38) and phosphorylated extracellular signal-regulated kinase (p-ERK)1/2 were detected by western blot analysis. The results from western blot analysis revealed that treatment with icariin decreased the levels of p-p38 and increased the levels of p-ERK1/2 in the IL-1β-stimulated SW1353 cells. In addition, treatment with icariin decreased the levels of RANK and RANKL. Furthermore, the suppressive effects of icariin on OPG and OPG/RANKL were greater than those exhibited by the p38 signaling pathway inhibitor (SB203580). The findings of the the present study suggest that icariin has therapeutic potential for use in the treatment of OA.

Influence of Calcium Hydroxide–loaded Microcapsules on Osteoprotegerin and Receptor Activator of Nuclear Factor Kappa B Ligand Activity

IntroductionCalcium hydroxide (Ca[OH]2) microcapsules were synthesized to allow controlled release of Ca(OH)2. The aim of this study was to evaluate the influence of Ca(OH)2 microcapsules on osteoprotegerin (OPG) activity, receptor activator of nuclear factor kappa B ligand (RANKL) activity, and the OPG/RANKL ratio compared with pure Ca(OH)2 powder and Vitapex (Neo Dental Chemical Products Co Ltd, Tokyo, Japan). MethodsOne formula of Ca(OH)2 microcapsules was evaluated, and pure Ca(OH)2 powder was used as a control. A commonly used Ca(OH)2 medication containing an oily vehicle (Vitapex) was also evaluated, and the in vitro release profile of Vitapex was studied. The human osteosarcoma cell line MG63 was used to evaluate the influence of Ca(OH)2 microcapsules, pure Ca(OH)2 powder, and Vitapex on OPG and RANKL activity. The relative messenger RNA (mRNA) expression of OPG and RANKL was determined by real-time polymerase chain reaction. The protein expression of OPG and RANKL in supernatants was measured using enzyme-linked immunosorbent assay. ResultsVitapex prolonged the release of Ca(OH)2 compared with pure Ca(OH)2 powder, and the release rate of Vitapex was faster than that of the microcapsules. The OPG/RANKL ratio in the microcapsules group was up-regulated at both the mRNA and protein levels compared with the negative control group and the pure Ca(OH)2 powder group. The ratio in the Vitapex group was lower than the microcapsule group both at the mRNA and protein levels. ConclusionsCa(OH)2 microcapsules increased the expression of OPG although they did not increase the expression of RANKL compared with pure Ca(OH)2 powder and Vitapex. This increase in expression led to an increase in the OPG/RANKL ratio and eventual inhibition of osteoclast activity.

Influences of IL-6R antibody on PMMA bone cement-mediated expression of OPG and RANKL in synovial fibroblasts

Effect of interleukin-6 receptor (IL-6R) antibody on polymethyl methacrylate (PMMA) bone cement-mediated expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL) in synovial fibroblasts was investigated. Synovial tissue obtained from total knee arthroplasty was digested and cultured. Inverted microscope was employed to observe the synovial cells and immunocytochemistry (SABC method) staining was used to identify synovial fibroblasts. This experiment was divided into three groups according to different culture media: PMMA group (75 μg/mL PMMA bone cement particles), IL-6R antibody group (10 ng/mL IL-6R antibody+75 μg/mL PMMA bone cement particles), and control group (no IL-6R antibody or PMMA bone cement particles). Influence of IL-6R antibody and PMMA on proliferation of synovial fibroblasts was measured by cell counting kit-8 (CCK-8). ELISA method was used to measure OPG and RANKL levels in culture solution. Fluorescence quantitative real-time PCR (FQ-PCR) was used to detect the expression of OPG and RANKL mRNA. After three consecutive passages, more than 95% of the primary synovial cells became long spindle fibroblast-like cells. SABC staining results showed that the fibroblast-like cells were negative for anti-CD68 antibody and positive for anti-vimentin antibody, with brown madder stained. CCK-8 test demonstrated that the absorbance (A) value at 450 nm was significantly lower in IL-6R antibody group than in PMMA group and control group (P<0.01), but there was no statistically significant difference in A value at 450 nm between the control group and PMMA group (P>0.05). Results of ELISA indicated that the expression of OPG was significantly higher in IL-6R antibody group than in PMMA group and control group (P<0.01). The expression of RANKL was inhibited (P<0.05), and the ratio of OPG/RANKL was significantly increased in IL-6R antibody group as compared with PMMA group and control group. There was no significant difference in the expression of OPG between control group and PMMA group (P>0.05), but the expression of RANKL was higher in PMMA group than in control group (P<0.05), and there was a significant difference in the ratio of OPG/RANKL between them (P<0.05). Results of FQ-PCR revealed the expression of RANKL mRNA was significantly inhibited (P<0.01) and the expression of OPG mRNA was significantly increased (P<0.01) in IL-6R antibody group as compared with PMMA group and control group. The expression of RANKL mRNA was higher in PMMA group than in control group (P<0.05), but the expression of OPG mRNA had no significant difference between them (P>0.05). IL-6R antibody could significantly increase the expression of OPG, but inhibit the expression of RANKL, which might provide a theoretical basis of molecular biology for the prevention and treatment of aseptic loosening of prosthesis.

Effects of exercise combined with alendronate sodium tablets on RANKL/OPG balance and MAPK signaling pathways in ovariectomized rats

Objective To observe the effect of exercise combined with alendronate sodium tablets(Aln)on receptor activator of nuclear factor kappa B ligand(RANKL)/osteoprotegerin(OPG)expression and mitogen-activated protein kinase (MAPK)signaling pathways in ovariectomized(OVX)rats.Methods A total of 90 6-month-old female SD rats were evenly randomized into sham group,OVX group,Aln treatment(OVX+Aln)group,exercise therapy(OVX+EX)group,and Aln combined with exercise treatment(OVX+Aln+EX)group.Eight weeks after ovariectomy,rats in the latter three groups were treated with intragastric 1mg/(kg·d)Aln and/or exercise.After 12weeks of treatment,dual-energy X-ray absorptiometry was used to examine the bone mineral density (BMD)of the left and right femurs.Enzyme-linked immunosorbent assay (ELISA)was used to determine the serum levels of RANKL and OPG.Real-time PCR was used to examine RANKLand OPG mRNA expression in bone tissues.Western blotting analysis was used to examine the phosphorylation of c-Jun N-terminal kinase(JNK),p38 MAPK (p38),extracellular-regulated kinase(ERK),and c-Fos protein expression in bone tissues.Results The following results were observed after 12-week treatment.(1)Compared with sham group,the BMD values of the left and right femurs were significantly decreased in OVX group (P0.05).The serum RANKL level was significantly increased,serum OPG level was significantly decreased,and the RANKL/OPG ratio was significantly increased (all P0.05).The RANKL mRNA expression was significantly increased and OPG mRNA expression was significantly decreased in bone tissues(all P0.05).(2)Compared with OVX group,the BMD values of left and right femurs of OVX+ Aln,OVX+EX and OVX+Aln+EX groups were significantly increased (all P0.05).The serum RANKL level was significantly decreased,serum OPG level was significantly increased,and the RANKL/OPG ratio decreased(all P0.05).The RANKL mRNA expression was significantly decreased,and OPG mRNA expression was significantly increased in bone tissues (all P0.05).(3)The femoral BMD values,serum RANKLand OPG levels,and RANKL and OPG mRNA expression in bone tissues in OVX+Aln+EX group were significantly different from those in OVX+Aln group and OVX+EX group(all P 0.05).(4)Compared with sham group,bone tissue p-JNK and c-Fos expression in OVX group was significantly increased (all P0.05).However,compared with OVX group,bone tissue p-JNK and c-Fos expression in OVX+Aln,OVX+EX and OVX+Aln+EX groups was significantly decreased(all P0.05),and there were no significant differences among the latter three groups.p-p38and p-ERK expression was similar in all groups.Conclusion Exercise combined with Aln can improve the BMD values of left and right femurs in OVX rats,which may be related to the regulation of RANKL/OPG balance and inhibition of JNK phosphorylation and c-Fos expression.

287: Th17-producing IL-17 on periodontal ligament induced root resorption in mouse models of rheumatoid arthritis and atopic dermatitis

Objective The aim of this study was to investigate how T-helper (Th) 17 cells and interleukin (IL)-17 contribute to root resorption during orthodontic tooth movement. Materials and methods: Experiment 1: Rheumatoid arthritis (RA) model mice and wild type mice were subjected to the orthodontic force (OF) to induce root resorption (heavy OF) of the upper first molars. The double-immunofluorescence analysis for IL-17/CD4 was performed to detect Th17 cells in the periodontal ligament (PDL). Experiment 2: Atopic dermatitis (AD) model mice and wild type mice were subjected to the heavy OF to induce movement of the upper first molars. The expression levels of the tartrate-resistant acid phosphatase (TRAP), IL-17, IL-6, and receptor activator of nuclear factor kappaB ligand (RANKL) proteins were determined in the PDL by an immunohistochemical analysis. Moreover, the fluorescent localization immunoassay was performed to detect Th17 cells. Furthermore, the effects of the compression force on co-cultures of CD4 + cells from AD patients or healthy individuals and human PDL cells were investigated with regard to the levels of secretion and mRNA expression of IL-17, IL-6, RANKL, and osteoprotegerin (OPG). Results: Experiment 1: The double-immunofluorescence analysis for IL-17/CD4 detected immunoreaction. Experiment 2: The immunoreactivities for TRAP, IL-17, IL-6, and RANKL in the AD group were found to be significantly increased. The double-immunofluorescence analysis for IL-17/CD4 detected immunoreaction. The secretion of IL-17, IL-6 and RANKL, and the mRNA levels of IL-6 and RANKL in the AD patients were increased compared with those in healthy individuals. Conclusion: Th17-producing IL-17 may be associated with the deterioration of orthodontic root resorption.

284: Caspase-8-independent role of c-FLIP in Inflammasome Activation

Cellular FLICE inhibitory protein (c-FLIP) is an inhibitor of caspase-8 and is required for macrophage development. Recent studies reveal a selective role of caspase-8 in non-canonical IL-1β production that is independent of caspase-1 or inflammasome. Here we demonstrated that c-FLIP is an unexpected contributor to canonical inflammasome activation for the generation of caspase-1 and active IL-1β. Lipopolysaccharides (LPS)-, R848-, and heat-killed Listeria monocytogenes (HKLM)-induced IL-1β production in macrophages was attenuated in the absence of c-FLIP. Decreased IL-1β expression was attributed to a reduced activation of caspase-1 in c-FLIP-deficient cells. In contrast, the production of TNF- α was not affected by deficiency in c-FLIP. c-FLIP interacted with NLRP3 or procaspase-1. Proximity ligation assay revealed that c-FLIP is required for the full NLRP3 inflammasome assembly, and c-FLIP associates with NLRP3 inflammasome components in situ . c-FLIP-deficiency also reduced AIM2 inflammasome activation. In contrast, c-FLIP inhibited SMAC mimetic- or FasL-induced IL-1β generation that is caspase-8-mediated. Our results demonstrate a caspase-8-independent role of c-FLIP in the optimal activation of the NLRP3 and AIM2 inflammasomes, and suggest that c-FLIP could be a valid target for treatment of inflammatory diseases caused by over-activation of inflammasomes. The aim of this study was to investigate how T-helper (Th) 17 cells and interleukin (IL) −17 contribute to root resorption during orthodontic tooth movement. Experiment 1: Rheumatoid arthritis (RA) model mice and wild type mice were subjected to the orthodontic force (OF) to induce root resorption (heavy OF) of the upper first molars. The double-immunofluorescence analysis for IL-17/CD4 was performed to detect Th17 cells in the periodontal ligament (PDL). Experiment 2: Atopic dermatitis (AD) model mice and wild type mice were subjected to the heavy OF to induce movement of the upper first molars. The expression levels of the tartrate-resistant acid phosphatase (TRAP), IL-17, IL-6, and receptor activator of nuclear factor kappaB ligand (RANKL) proteins were determined in the PDL by an immunohistochemical analysis. Moreover, the fluorescent localization immunoassay was performed to detect Th17 cells. Furthermore, the effects of the compression force on co-cultures of CD4 + cells from AD patients or healthy individuals and human PDL cells were investigated with regard to the levels of secretion and mRNA expression of IL-17, IL-6, RANKL, and osteoprotegerin (OPG). Results: Experiment 1: The double-immunofluorescence analysis for IL-17/CD4 detected immunoreaction. Experiment 2: The immunoreactivities for TRAP, IL-17, IL-6, and RANKL in the AD group were found to be significantly increased. The double-immunofluorescence analysis for IL-17/CD4 detected immunoreaction. The secretion of IL-17, IL-6 and RANKL, and the mRNA levels of IL-6 and RANKL in the AD patients were increased compared with those in healthy individuals. Th17-producing IL-17 may be associated with the deterioration of orthodontic root resorption.