RANK/RANKL axis promotes migration, invasion, and metastasis of osteosarcoma via activating NF-κB pathway.

Osteosarcoma (OS) is one of the most prevalent primary bone tumors with a high degree of metastasis and poor prognosis. Epithelial-to-mesenchymal transition (EMT) is a cellular mechanism that contributes to the invasion and metastasis of cancer cells, and OS cells have been reported to exhibit EMT-like characteristics. Our previous studies have shown that the interaction between tumor necrosis factor superfamily member 11 (TNFRSF11A; also known as RANK) and its ligand TNFSF11 (also known as RANKL) promotes the EMT process in breast cancer cells. However, whether the interaction between RANK and RANKL enhances aggressive behavior by inducing EMT in OS cells has not yet been elucidated. In this study, we showed that the interaction between RANK and RANKL increased the migration, invasion, and metastasis of OS cells by promoting EMT. Importantly, we clarified that the RANK/RANKL axis induces EMT by activating the nuclear factor-kappa B (NF-κB) pathway. Furthermore, the NF-κB inhibitor dimethyl fumarate (DMF) suppressed migration, invasion, and EMT in OS cells. Our results suggest that the RANK/RANKL axis may serve as a potential tumor marker and promising therapeutic target for OS metastasis. Furthermore, DMF may have clinical applications in the treatment of lung metastasis in patients with OS.

Breaking Osteoclast-Acid Vicious Cycle to Rescue Osteoporosis via an Acid Responsive Organic Framework-Based Neutralizing and Gene Editing Platform.

In the osteoporotic microenvironment, the acidic microenvironment generated by excessive osteoclasts not only causes irreversible bone mineral dissolution, but also promotes reactive oxygen species (ROS) production to induce osteoblast senescence and excessive receptor activator of nuclear factor kappa-B ligand (RANKL) production, which help to generate more osteoclasts. Hence, targeting the acidic microenvironment and RANKL production may break this vicious cycle to rescue osteoporosis. To achieve this, an acid-responsive and neutralizing system with high in vivo gene editing capacity is developed by loading sodium bicarbonate (NaHCO3) and RANKL-CRISPR/Cas9 (RC) plasmid in a metal-organic framework. This results showed ZIF8-NaHCO3@Cas9 (ZNC) effective neutralized acidic microenvironment and inhibited ROS production . Surprisingly, nanoparticles loaded with NaHCO3 and plasmids show higher transfection efficiency in the acidic environments as compared to the ones loaded with plasmid only. Finally, micro-CT proves complete reversal of bone volume in ovariectomized mice after ZNC injection into the bone remodeling site. Overall, the newly developed nanoparticles show strong effect in neutralizing the acidic microenvironment to achieve bone protection through promoting osteogenesis and inhibiting osteolysis in a bidirectional manner. This study provides new insights into the treatment of osteoporosis for biomedical and clinical therapies.

Farrerol suppresses osteoclast differentiation and postmenopausal osteoporosis by inhibiting the nuclear factor kappa B signaling pathway.

Excessive bone resorption caused by upregulated osteoclast activity is a key factor in osteoporosis pathogenesis. Farrerol is a typical natural flavanone and exhibits various pharmacological actions. However, the role and mechanism of action of farrerol in osteoclast differentiation regulation remain unclear. This study aimed to evaluate the effects and mechanism of farrerol on the inhibition of osteoclastogenesis. Tartrate-resistant acid phosphatase staining, F-actin staining, and the pit formation assay were performed to examine the differentiation and functions of osteoclasts in vitro. The expression of proteins associated with the nuclear factor kappa B and mitogen-activated protein kinase signaling pathways was analyzed by western blotting. Dual X-ray absorptiometry, microcomputed tomography, and histopathological and immunohistochemical analyses were performed to determine the therapeutic effect of farrerol in vivo bone loss prevention. The effects of farrerol on osteoblastic bone formation were assessed using alkaline phosphatase, alizarin red S staining, and calcein-alizarin red S double labeling. Farrerol inhibited osteoclastogenesis and bone resorption in osteoclasts by suppressing nuclear factor kappa B signaling rather than mitogen-activated protein kinase signaling in vitro. Farrerol protected mice against ovariectomy-induced bone loss by inhibiting osteoclast-mediated bone resorption, instead of promoting osteoblast-mediated bone formation in vivo. The findings of the current study revealed that farrerol is a potential therapeutic agent for osteoporosis.

Extracellular Signal-Regulated Kinases Play Essential but Contrasting Roles in Osteoclast Differentiation.

Bone homeostasis is regulated by the balanced actions of osteoblasts that form the bone and osteoclasts (OCs) that resorb the bone. Bone-resorbing OCs are differentiated from hematopoietic monocyte/macrophage lineage cells, whereas osteoblasts are derived from mesenchymal progenitors. OC differentiation is induced by two key cytokines, macrophage colony-stimulating factor (M-CSF), a factor essential for the proliferation and survival of the OCs, and receptor activator of nuclear factor kappa-B ligand (RANKL), a factor for responsible for the differentiation of the OCs. Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs), p38, and c-Jun N-terminal kinases, play an essential role in regulating the proliferation, differentiation, and function of OCs. ERKs have been known to play a critical role in the differentiation and activation of OCs. In most cases, ERKs positively regulate OC differentiation and function. However, several reports present conflicting conclusions. Interestingly, the inhibition of OC differentiation by ERK1/2 is observed only in OCs differentiated from RAW 264.7 cells. Therefore, in this review, we summarize the current understanding of the conflicting actions of ERK1/2 in OC differentiation.

[Expression profile of circular RNA in inflammatory response in human bronchial epithelial cells induced by carbon black nanoparticles].

Objective: To explore the toxic effect of carbon black nanoparticles on human bronchial epithelial cells, and identify the differentially expressed circular RNA based on the full transcriptome high-throughput sequencing, so as to provide evidence for the development of biomarkers exposed to carbon black nanoparticles and their application on epigenetic toxicology. Methods: In June 2020, 16 HBE cells were treated with carbon black nanoparticles at concentrations of 20, 40 and 80 µg/ml, and 16 HBE cells without any intervention were used as the control group. The cytotoxicity of carbon black nanoparticles was detected by CCK8 and LDH experiments. Real-time quantitative fluorescent PCR (qRT-PCR) and ELISA were used to detect the changes of interleukin-6 (IL-6) and interleukin-8 (IL-6, IL-8) mRNA and protein levels of carbon black nanoparticles with concentration gradient after 72 h exposure. Western blot analysis was conducted to detect the expression levels of toll-like receptor 4 (TLR4), phosphorylated nuclear factor-κB (P-NF-κB), apoptosis-related speckled protein (ASC) and Caspase-1 associated with nuclear factor-κB. According to high-throughput sequencing results, differentially expressed Circrnas were screened and identified by qRT-PCR, and those with stable differentially expressed circrnas and the strongest association with the NF-κB pathway were selected for ring performance identification. Results: After being exposed to carbon black nanoparticles for 72 h, the activity of 16HBE cells decreased significantly (P<0.05), and the release of lactate dehydrogenase increased significantly (P<0.05). Compared with control group, mRNA expression levels of IL-6 and IL-8, protein levels of IL-6 and IL-8 were increased, and protein levels of TLR4, p-NF-κB, ASC and Caspase-1 were significantly up-regulated in 16 HBE cells of different concentrations, with statistical significance (P<0.05). Compared with the control group, a total of 492 differentially expressed circular Rnas (|log2 FC|>1) were detected. Among the 5 differentially expressed (P<0.05) circular Rnas, circ_002642 was selected as the object of subsequent research on circular Rnas, affter 72 hours of exposure to 80 µg/ml CBNPs, 16HBE cells showed signlficantly higher expression of circ_002642 (P<0.05) . Conclusion: Carbon black nanoparticles can induce differentially expressed circular RNAs associated with inflammatory response in human bronchial epithelial cells.

Impact of the Rapid Normalization of Chronic Hyperglycemia on the Receptor Activator of Nuclear Factor-Kappa B Ligand and the Osteoprotegerin System in Patients Living with Type 2 Diabetes: RANKL-GLYC Study.

The RANKL-GLYC study aims to explore the impact of the rapid correction of chronic hyperglycemia on the receptor activator of nuclear factor-kappa B ligand (RANKL) and its antagonist osteoprotegerin (OPG). RANKL and OPG are considered the main factors in the pathophysiology of Charcot neuroarthropathy, a devastating complication of the joints that remains poorly understood. The study began recruiting patients in September 2021 and ends in June 2022; the final study results are scheduled for January 2023.

Pathophysiological mechanisms of root resorption after dental trauma: a systematic scoping review.

BACKGROUND: The objective of this scoping review was to systematically explore the current knowledge of cellular and molecular processes that drive and control trauma-associated root resorption, to identify research gaps and to provide a basis for improved prevention and therapy. METHODS: Four major bibliographic databases were searched according to the research question up to February 2021 and supplemented manually. Reports on physiologic, histologic, anatomic and clinical aspects of root resorption following dental trauma were included. Duplicates were removed, the collected material was screened by title/abstract and assessed for eligibility based on the full text. Relevant aspects were extracted, organized and summarized. RESULTS: 846 papers were identified as relevant for a qualitative summary. Consideration of pathophysiological mechanisms concerning trauma-related root resorption in the literature is sparse. Whereas some forms of resorption have been explored thoroughly, the etiology of others, particularly invasive cervical resorption, is still under debate, resulting in inadequate diagnostics and heterogeneous clinical recommendations. Effective therapies for progressive replacement resorptions have not been established. Whereas the discovery of the RANKL/RANK/OPG system is essential to our understanding of resorptive processes, many questions regarding the functional regulation of osteo-/odontoclasts remain unanswered. CONCLUSIONS: This scoping review provides an overview of existing evidence, but also identifies knowledge gaps that need to be addressed by continued laboratory and clinical research.

Effects of photobiomodulation at different wavelengths on orthodontically induced root resorption in orthodontic retention period: a micro-CT and RT-PCR study.

The aim of this study was to evaluate and compare the reparative and inhibitory effects of single wavelength photobiomodulation (SW-PBM) and of cumulative increased wavelength photobiomodulation (CW-PBM) on orthodontically induced inflammatory root resorption (OIIRR). Thirty-three Wistar albino rats were divided into five groups: untreated group (negative control), only relapse group (positive control-1), only retention group (positive control-2), SW-PBM group (650 nm, 100 mW/cm2), and CW-PBM group (532-650-940 nm, 100 mW/cm2). Orthodontic tooth movement was induced experimentally in rats for 10 days with an applied force of 50 cN; retention and therapeutic approaches were performed concurrently. At the end of the experiment, maxillary quadrants were prepared for micro-CT analysis and real-time polymerase chain reaction (RT-PCR) analysis. After the Shapiro-Wilk normality test, Kruskal-Wallis test followed post hoc Bonferroni test and paired samples t test was used for statistical evaluation of the data. Resorption lacunae volume (p < 0.001), number of resorption lacunae (p < 0.05), and percentage of the resorption (PR) lacunae (p < 0.001) decreased with PBM applications when compared with the positive control groups, and the mean PR was similar in the negative control group when compared with SW-PBM group. Receptor activator of nuclear factor kappa B ligand (RANKL) levels of the PBM groups were lower (p < 0.05) than those of the positive control groups. Cyclooxygenase-2 (COX-2) expression levels significantly decreased with PBM administration (p < 0.05). No significant change was found in osteoprotegerin (OPG) expression levels and OPG/RANKL ratios (p > 0.05). PBM applications showed marked inhibitory and reparative effects on OIIRR by modulating the RANKL and COX-2 expression levels. However, the effects of the different wavelengths were similar to each other.Graphical abstract.

Finely-Tuned Calcium Oscillations in Osteoclast Differentiation and Bone Resorption.

Calcium (Ca2+) plays an important role in regulating the differentiation and function of osteoclasts. Calcium oscillations (Ca oscillations) are well-known phenomena in receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption via calcineurin. Many modifiers are involved in the fine-tuning of Ca oscillations in osteoclasts. In addition to macrophage colony-stimulating factors (M-CSF; CSF-1) and RANKL, costimulatory signaling by immunoreceptor tyrosine-based activation motif-harboring adaptors is important for Ca oscillation generation and osteoclast differentiation. DNAX-activating protein of 12 kD is always necessary for osteoclastogenesis. In contrast, Fc receptor gamma (FcRγ) works as a key controller of osteoclastogenesis especially in inflammatory situation. FcRγ has a cofactor in fine-tuning of Ca oscillations. Some calcium channels and transporters are also necessary for Ca oscillations. Transient receptor potential (TRP) channels are well-known environmental sensors, and TRP vanilloid channels play an important role in osteoclastogenesis. Lysosomes, mitochondria, and endoplasmic reticulum (ER) are typical organelles for intracellular Ca2+ storage. Ryanodine receptor, inositol trisphosphate receptor, and sarco/endoplasmic reticulum Ca2+ ATPase on the ER modulate Ca oscillations. Research on Ca oscillations in osteoclasts has still many problems. Surprisingly, there is no objective definition of Ca oscillations. Causality between Ca oscillations and osteoclast differentiation and/or function remains to be examined.

Aliskiren, tadalafil, and cinnamaldehyde alleviate joint destruction biomarkers; MMP-3 and RANKL; in complete Freund's adjuvant arthritis model: Downregulation of IL-6/JAK2/STAT3 signaling pathway.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease, which is accompanied by progressive joint damage and disability. The intolerability of conventional antirheumatic drugs by some patients necessitates the search for effective antirheumatic agents having better tolerability. In the current work, we aimed to investigate the efficacy of cinnamaldehyde, tadalafil, and aliskiren as potential antirheumatic candidates and to explore their modulatory effects on joint destruction, inflammatory response, and intracellular signaling. Arthritis was induced in female Wistar rats by complete Freund's adjuvant (CFA) 0.4 ml s.c. on days 1, 4, and 7. Treated groups received their respective drugs, starting from day 13, daily for 3 weeks. Methotrexate and prednisolone were the standard antirheumatic drugs, while cinnamaldehyde, tadalafil, and aliskiren were the test agents. Treatment with cinnamaldehyde, tadalafil, or aliskiren reduced serum levels of rheumatoid factor, and pro-inflammatory cytokines; tumor necrosis factor-alpha and interleukin-6 (IL-6), along with elevated level of IL-10 which is an anti-inflammatory cytokine. Besides, cartilage and bone destruction biomarkers; matrix metalloproteinase-3 (MMP-3) and receptor activator of nuclear factor-kappa B ligand (RANKL); were significantly reduced after treatment with the test agents, which was further confirmed by histopathological investigation. The elevated protein expressions of phosphorylated-Janus kinase 2 (p-JAK2), phosphorylated-signal transducer and activator of transcription 3 (p-STAT3), and inducible nitric oxide synthase (iNOS) in articular tissue were markedly attenuated after treatment with cinnamaldehyde, tadalafil, or aliskiren, while that of endothelial nitric oxide synthase (eNOS) was greatly enhanced. In addition, oxidative stress and inflammatory markers such as malondialdehyde, nitric oxide, and myeloperoxidase were reduced in joint tissue after treatment with the test agents, while glutathione content was elevated. Furthermore, the renin inhibitor aliskiren produced effects close to those of the normal and methotrexate, the gold standard antirheumatic drug, in most of the measured parameters. Collectively, these findings led to the assumption that the downregulation of IL-6/JAK2/STAT3 signaling by cinnamaldehyde, tadalafil, and aliskiren could alleviate joint destruction by MMP-3 and RANKL, reduce iNOS, and enhance eNOS expressions. Moreover, aliskiren could be a promising therapeutic agent for RA, because of its ability to normalize most of the measured parameters after CFA-induced arthritis.

MiRNA-199a-5p positively regulated RANKL-induced osteoclast differentiation by target Mafb protein.

MicroRNAs are involved in osteoclast differentiation. Although miR-199a-5p plays an important role in many different systems and diseases, its function during osteoclastogenesis remains unclear. In this study, we investigated the function and the target gene of miR-199a-5p in osteoclast differentiation. The in vitro data showed that miR-199a-5p was significantly upregulated after the stimulation by receptor activator of nuclear factor kappa-B ligand in macrophages and RAW 264.7 cells. After transfection of miR-199a-5p mimic, the messenger RNA expression level of nuclear factor of activated T-cells cytoplasmic 1, tartrate-resistant acid phosphatase (TRAP), and receptor activator of nuclear factor kappa-B was significantly increased in RAW 264.7 cells and the number of TRAP-positive cells was also increased. MiR-199a-5p inhibitor showed the complete opposite outcome which brought additional proof to our finding. Overexpression of miR-199a-5p led to downregulation of Mafb protein. The luciferase activity was obviously repressed when WT-pGL3-Mafb and miR-199a-5p mimics were cotransfected into 293 T cells and the inhibitors cotransfected demonstrated reverse result. MiR-199a-5p overexpressed during osteoclast differentiation and positively regulated osteoclast formation in vitro by target Mafb.

Accumulation of hyaluronic acid in stromal cells modulates osteoclast formation by regulation of receptor activator of nuclear factor kappa-B ligand expression.

Hyaluronic acid (HA) has a pivotal role in bone and cartilage metabolism. In this study, we investigated the effect and underlying mechanisms of HA accumulation on the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) induced by 1α,25(OH)2D3 and dexamethasone in stromal cells, which support osteoclastogenesis. Degradation of HA by hyaluronidase (HA'ase) treatment enhanced the expression of RANKL in ST2 cells stimulated with 1α,25(OH)2D3 and dexamethasone. Down-regulation of hyaluronan synthase 2 (HAS2) expression by siRNA also stimulated RANKL expression induced by 1α,25(OH)2D3 and dexamethasone. Results from a cell co-culture system with bone marrow cell showed that 1α,25(OH)2D3 and dexamethasone-induced RANKL expression in HA'ase treated- and HAS2 siRNA transfected-ST2 cells was down-regulated by treatment of cells with high molecular weight HA. In contrast, transforming growth factor-ß1 (TGF-ß1), which stimulates HAS2 expression and HA synthesis, down-regulated RANKL expression induced by 1α,25(OH)2D3 and dexamethasone. Interestingly, knockdown of has2 gene enhanced the expression of vitamin D receptor (VDR) and phosphorylation of signal transducers and activator of transcription 3 (STAT3) in ST2 cells stimulated by 1α,25(OH)2D3 and dexamethasone. These results indicate that accumulation of HA in bone marrow cells may affect RANKL-mediated osteoclast-supporting activity via regulation of VDR and STAT3 signaling pathways.

Effect of technetium-99 conjugated with methylene diphosphonate (99 Tc-MDP) on OPG/RANKL/RANK system in vitro.

BACKGROUND: RANKL and RANK play an important role in jaw resorption during the development of the ameloblastomas. Therefore, the aim of this study was to explore the effect of 99 Tc-MDP on OPG/RANKL/RANK system on RAW264.7 and MC3T3-E1 cell lines in vitro and provide the theoretical basis for the clinical treatment of the jaw ameloblastoma. METHODS: Different concentrations of 99 Tc-MDP were used to treat RAW264.7 and MC3T3-E1 cell lines. The cell proliferative inhibition rate was analyzed by CCK-8. Cell apoptosis and cell cycle were detected by flow cytometry. Western blot was used to detect the expression of OPG, RANKL, and RANK. RESULTS: Treatment of RAW264.7 cell lines with different concentrations of 99 Tc-MDP had inhibitory effects and decreased the expression of RANK protein. The cell proliferation of 99 Tc-MDP on MC3T3-E1 cell lines was stronger at 48 hours than at 24 hours except for 100 µg/mL concentration group. Compared with the concentration of 0.01 µg/mL, the treatment of MC3T3-E1 cells with 100 µg/mL 99 Tc-MDP showed that the cell proliferative effect decreased at 24 hours and 48 hours (P < 0.05). After treatment with 0.01 µg/mL 99 Tc-MDP, the expression of OPG in MC3T3-E1 cells was significantly increased (P < 0.05). Compared with 0.01 µg/mL, the expression of RANKL was decreased after treatment with 100 µg/mL 99 Tc-MDP (P < 0.05). CONCLUSION: 99 Tc-MDP can induce apoptosis of RAW264.7 cells and inhibit the expression of RANK protein. The effect of 0.01 µg/mL of low concentration of 99 Tc-MDP can promote the proliferation of MC3T3-E1 cells and increase the expression of OPG and RANKL protein. 99 Tc-MDP may have adjuvant therapeutic effects on the treatment of jaw ameloblastoma.

Association of OPG-RANKL ratio with left ventricular hypertrophy and geometric remodeling in male overweight/obese youths.

PURPOSE: Receptor activator of nuclear factor kappa B ligand/receptor activator of nuclear factor kappa B/osteoprotegerin (RANKL/RANK/OPG) axis has been hypothesized as a potential mediator of left ventricular hypertrophy (LVH). The aim of the study was to assess whether circulating concentrations of RANKL, RANK, and OPG were associated with early signs of morphological cardiac changes in overweight/obese youths. METHODS: We determined serum levels of RANKL, RANK and OPG by enzyme-linked immunosorbent assays in 188 overweight/obese children and adolescents. LV mass index (LVMI) and relative wall thickness (RWT) were estimated using M-mode echocardiography. RESULTS: OPG and RANKL levels were higher among girls than among boys [1.73 (1.64-1.86) and 3.28 (1.90-6.37) pmol/L, respectively, vs. 1.69 (1.59-1.82) and 2.12 (1.52-3.80) pmol/L; p = 0.02 and p = 0.0001, respectively], but the OPG/RANKL ratio was lower [0.52 (0.26-0.88) vs 0.77 (0.44-1.11); p = 0.001]. In gender-specific multivariate linear regression, OPG/RANKL ratio was associated with LVMI and RWT in boys but not in girls. In multiple logistic regression, after adjustment for clinical variables, OPG/RANKL ratio was associated with concentric remodeling, eccentric and concentric LVH in boys but not in girls. CONCLUSION: OPG/RANKL ratio is independently associated with LVH and patterns of LV structural remodeling in male overweight/obese children and adolescents.

Local injection of RANKL facilitates tooth movement and alveolar bone remodelling.

OBJECTIVES: To investigate the effect of local injection of receptor activator of nuclear factor kappa B ligand (RANKL) on experimental tooth movement and subsequent alveolar bone remodelling in mice. MATERIALS AND METHODS: Sixty mice were randomised to receive daily local RANKL or phosphate-buffered saline injections in the buccal premaxillary bone for 14 of 21 days of incisor movement, followed by a 21-day retention period. Five mice from each group were euthanised on days 0, 3, 7, 14, 21 and 42, and specimens were prepared for haematoxylin and eosin, tartrate-resistant acid phosphatase and immunohistochemical staining. Five mice from each group were subjected to serial microcomputed tomography until day 42 for tooth movement and bone volume quantification. RESULTS: The experimental group showed significantly greater tooth movement and bone volume reduction on days 14 and 21; an increased osteoclast number on days 3, 7, 14 and 21; and no difference on day 42. Higher RANKL expression was observed on days 7 and 14, with remarkable alkaline phosphatase activity. No significant systemic changes were observed. CONCLUSION: Local RANKL injection leads to increased osteoclastic activity and facilitates tooth movement, followed by subsequent alveolar bone formation; this implies a reversible transitional acceleration of bone resorption.

Expression of the RANK/RANKL/OPG system in the human intervertebral disc: implication for the pathogenesis of intervertebral disc degeneration.

BACKGROUND: The expression of the receptor activator of nuclear factor kappa B (RANK) /RANK ligand (RANKL) /osteoprotegerin (OPG) system and its association with the progression of intervertebral disc (IVD) degeneration has recently been reported in a human IVD. However, the effect of the RANK/RANKL/OPG system on the matrix metabolism of human IVD cells, especially on the expression of catabolic factors relevant to IVD degeneration, remains unknown. The purpose of this study was to examine the expression of the RANK/RANKL/OPG system, and then to evaluate the effect of this system on the expression of catabolic factors by human IVD cells. METHODS: Annulus fibrosus (AF) and nucleus pulposus (NP) cells isolated by sequential enzyme digestion from human IVD tissues obtained during spine surgeries were monolayer cultured. The expression of the RANK/RANKL/OPG system was determined using immunohistochemical methods and real-time polymerase chain reaction (PCR). To evaluate the influence of interleukin-1 beta (IL-1ß) stimulation on the mRNA expression of RANK, RANKL, and OPG, recombinant human IL-1ß (rhIL-1ß) was administered in the culture media of IVD cells. To examine the influence of RANKL signaling on the expression of matrix metalloprotease-3 (MMP-3), MMP-13, and IL-1ß, the cells were cultured with exogenous recombinant human RANKL (rhRANKL), recombinant human OPG (rhOPG) or anti-human RANKL mouse monoclonal antibody (ahRANKL-mAB) with or without rhIL-1ß. RESULTS: Immunoreactivity to RANK/RANKL/OPG and the mRNA expression of the three genes were obviously identified in both AF and NP cells. rhIL-1ß stimulation significantly upregulated the mRNA expression level of RANK/RANKL/OPG. The mRNA expression of catabolic factors was significantly upregulated by stimulation of rhRANKL in the presence of rhIL-1ß. On the other hand, the administration of either rhOPG or ahRANKL-mAB significantly suppressed the mRNA expression of catabolic factors that had been upregulated by rhIL-1ß stimulation. The suppressive effect of ahRANKL-mAB against rhIL-1ß stimulation was also confirmed by the protein expression of MMP-3. CONCLUSIONS: The present study showed that the RANK/RANKL/OPG system may be involved in the progression of IVD degeneration. This study also suggested the potential use of anti-RANKL monoclonal antibody and OPG as therapeutic agents to suppress the progression of IVD degeneration.

Expression Profiling of Receptor-Activator of Nuclear Factor-Kappa B Ligand in Soft Tissue Tumors.

Bone and soft tissue tumors are derived from mesenchymal cells, and they are hard to treat. Receptor-activator of nuclear factor-kappa B ligand (RANKL) is an essential cytokine for osteoclast differentiation and activation and is expressed on the surface of osteoblasts or stromal cells. In this study, to explore the potential of denosumab treatment for soft tissue tumors, we analyzed the expression profiles of RANKL mRNA in 425 tumor specimens of 33 histological types by real-time RT-PCR. Denosumab is a monoclonal antibody that prevents the binding of RANKL to receptor-activator of nuclear factor-kappa B (RANK). For comparison, the relative expression levels of RANK and osteoprotegerin (OPG) mRNAs were also measured. OPG functions as a soluble decoy receptor for RANKL. Higher expression levels of RANKL mRNA were detected in calcifying aponeurotic fibroma, fibrosarcoma, calcifying epithelioma, myositis ossificans, heterotopic calcification, giant cell tumor of the tendon sheath (GCTTS), and pigmented villonodular synovitis (PVNS), compared with the levels of other tumor types. Moreover, the expression levels of RANK mRNA were highest in GCTTS, followed by myositis ossificans and PVNS, whereas the expression levels of OPG mRNA were greatly varied among these histological types. We then analyzed RANKL protein expression by immunohistochemistry in 57 tumor specimens with higher expression levels of RANKL mRNA. RANKL-positive cells were detected in GCTTS, PVNS, myositis ossificans, heterotopic calcification, and calcifying aponeurotic fibroma. In conclusion, RANKL is expressed in subsets of soft tissue tumors with calcification, and denosumab is a potential therapeutic option for soft tissue tumors expressing RANKL.

Long term (4 years) improved insulin sensitivity following islet cell transplant in type 1 diabetes.

BACKGROUND: Impaired insulin sensitivity (IS) predicts complications and mortality in type 1 diabetes (T1D). Insulin sensitivity improves shortly after islet cell transplant for T1D, yet long-term changes in IS and associated factors such as patient characteristics, transplant factors, clinical management, and IS-related biomarkers are unknown. METHODS: Up to 9 years (mean 4) of longitudinal data were available on 22 adults (18 female) with T1D who received 1 to 3 transplants in Phase 1/2 or 3 clinical trials (2004-2014). Metabolic testing posttransplant estimated IS by the Homeostasis Model Assessment for Insulin Resistance (HOMA-IR; 111 observations) and the Simple Index of Insulin Sensitivity (SIis ; 95 observations). RESULTS: Simple Index of Insulin Sensitivity significantly increased the first year posttransplant (P = .02), then stabilized (P = .39); HOMA-IR remained stable posttransplant (P = .92). Adjusting for age and BMI, higher SIis was associated with lower HbA1c following transplant (P = .03). Greater IS as measured by lower HOMA-IR and higher SIis was associated with lower fasting C-peptide (both P ≤ .04) and also with higher exenatide dose (both P ≤ .01). More islets transplanted were associated with higher SIis (P < .0001). Lower leptin at transplant predicted lower HOMA-IR and higher SIis after transplant, and lower bone marker receptor activator of nuclear factor kappa-B ligand predicted lower HOMA-IR (all P ≤ .01). CONCLUSIONS: Insulin sensitivity measured by SIis was improved several years following transplant, while IS measured by HOMA-IR did not worsen. Higher exenatide dose, more islets transplanted, and diet and exercise (lowering leptin and receptor activator of nuclear factor kappa-B ligand) may improve IS, which may enhance glycaemic control and lower metabolic demand on transplanted islets. Long-term clamp studies are needed to confirm these results.

Study on site-specific expression of bone formation and resorption factors in human dental follicles.

We sought to investigate site-specific expression of bone-regulatory factors expressed by human dental follicles and to compare the stimulated expression of tumour necrosis factor (ligand) superfamily, member 11/tumour necrosis factor receptor superfamily, member 11b (RANKL/OPG) in human dental follicle cells (HDFCs) from different patients. Analysis of bone-regulatory markers in follicles from 12 different study participants was performed using RT-qPCR and immunofluorescence; apical and coronal segments from each dental follicle were processed independently. Four additional dental follicles were used for cell cultures; HDFCs were precultured in osteogenic medium to initiate differentiation and thereafter cultured with 10-6 M forskolin (FSK) to activate the protein kinase cAMP (PKA/cAMP) signalling pathway and induce RANKL/OPG expression. We demonstrate that RANKL expression is significantly higher in the coronal part of follicles than in the apical part. High levels of collagen type 1 (COL1), alkaline phosphatase (ALP) and Gap-junction protein, alpha 1, 43 kDa (CX43) were expressed, whereas expression of Sp7 transcription factor (OSX), bone morphogenetic protein 2 (BMP2), colony-stimulating factor 1 (CSF-1), chemokine (C-C motif) ligand 2 (MCP1), and OPG was low in all samples. The immunofluorescence localization of CSF-1, MCP1, osteocalcin (OCN), RANKL, and BMP2 was not specific for either part of the follicles. In conclusion, a consistently high expression of CX43 suggests that gap-junction communication in HDFCs is essential for the eruption process. Furthermore, the induced expression of RANKL in HDFCs varies significantly between individuals and may relate to clinical variations in tooth eruption.

Radix Paeoniae Rubra stimulates osteoclast differentiation by activation of the NF-κB and mitogen-activated protein kinase pathways.

BACKGROUND: Radix Paeoniae Rubra (RPR), a traditional Chinese herb, has anti-inflammatory and immuno-regulatory properties. This study explored the effects of RPR on stimulation of osteoclast differentiation in RAW264.7 cells and peripheral blood mononuclear cells (PBMC)s. METHODS: The mature osteoclasts were measured by bone resorption assays and TRAP staining. JNK, ERK, p38 and NF-κB inhibitors were used applied in order to verify their contribution in RPR-induced osteoclast differentiation. The NF-κB and MAPK pathways were evaluated by western blotting, RT-PCR and luciferase assay. RESULTS: RPR induced osteoclast differentiation in a dose-dependent manner and induced the resorption activity of osteoclasts differentiation of RAW264.7 cells and PBMCs. Western blotting showed that RPR treatment induced phosphorylation of JNK, ERK, and p38 in RAW 264.7 cells. Treatment of JNK, ERK, and p38 MAP kinase inhibitors verified the contribution of JNK, ERK and p38. RPR treatment induced c-Fos and NFATc1 protein expression; NF-κB inhibitor treatment and luciferase assay verified the contribution of the NF-κB pathway. CONCLUSIONS: This study demonstrated the interesting effect, in which RPR stimulated osteoclast differentiation in murine RAW264.7 cells and human monocytes.