Release Of RANKL Research Articles

Neutrophil extracellular traps mediate bone erosion in rheumatoid arthritis by enhancing RANKL-induced osteoclastogenesis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that can cause bone erosion due to increased osteoclastogenesis. Neutrophils involvement in osteoclastogenesis remains uncertain. Given that neutrophil extracellular traps (NETs) can act as inflammatory mediators in rheumatoid arthritis, we investigated the role of NETs in stimulating bone loss by potentiating osteoclastogenesis during arthritis. The level of NETs in synovial fluid from arthritis patients was assessed. Bone loss was evaluated by histology and micro-CT in antigen-induced arthritis (AIA)-induced WT mice treated with DNase or in Padi4-deficient mice (Padi4flox/flox LysMCRE ). The size and function of osteoclasts and the levels of RANKL and osteoprotegerin (OPG) released by osteoblasts that were incubated with NETs were measured. The expression of osteoclastogenic marker genes and protein levels were evaluated by qPCR and western blotting. To assess the participation of TLR4 and TLR9 in osteoclastogenesis, cells from Tlr4-/- and Tlr9-/- mice were cultured with NETs. Rheumatoid arthritis patients had higher levels of NETs in synovial fluid than osteoarthritis patients, which correlated with increased levels of RANKL/OPG. Moreover, patients with bone erosion had higher levels of NETs. Inhibiting NETs with DNase or Padi4 deletion alleviated bone loss in arthritic mice. Consistently, NETs enhanced RANKL-induced osteoclastogenesis that was dependent on TLR4 and TLR9 and increased osteoclast resorptive functions in vitro. In addition, NETs stimulated the release of RANKL and inhibited osteoprotegerin in osteoblasts, favouring osteoclastogenesis. Inhibiting NETs could be an alternative strategy to reduce bone erosion in arthritis patients.

Assessment of Pain Sensation and Release of RANKL In Gingival Crevicular Fluid Accompanying Laser Application In Orthodontic Treatment: A Randomized Controlled Clinical Trial

Assessment of Pain Sensation and Release of RANKL In Gingival Crevicular Fluid Accompanying Laser Application In Orthodontic Treatment: A Randomized Controlled Clinical Trial

Fibroblast-like Synoviocytes – Actors in Osteoimmunology

AbstractRheumatoid arthritis (RA) is an immune mediated inflammatory disease (IMID), characterized by chronic inflammation and irreversible bone loss. Studies have shown that fibroblast-like synoviocytes (FLS), a key cell population in the pathogenesis of RA, have an impact on balancing bone-forming osteoblasts and bone-destroying osteoclasts towards joint damage. Once activated, RA-FLS are able to destroy cartilage and subchondral bone through the release of RANKL, members of the metalloproteinase family and many more cytokines, chemokines and growth factors. Additionally, RA-FLS are responsible for the perpetuation and chronicity of the disease due the interaction with immune cells supporting the influx of T and B lymphocytes, monocytes, macrophages neutrophils and dendritic cells from the blood stream into the inflamed synovial tissue. In this review we highlight the direct and indirect impact of synovial fibroblasts in RA on joint damage and disease progression. Moreover, we describe mechanisms of synovitis and regulators of bone homeostasis in further inflammatory joint diseases such as ankylosing spondylitis (AS) and psoriatic arthritis (PsA) and compare them to RA.

High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro.

Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the proinflammatory cytokine, high mobility group box 1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43def ), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/M-CSF treated bone marrow cells. In bone marrow macrophages (BMMs), toll-like receptor 4 (TLR4) inhibition with LPS-RS, but not receptor for advanced glycation end products (RAGE) inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly affects differentiation by activating TLR4 in BMMs and RAGE in preosteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.

Possible link between FSH and RANKL release from adipocytes in men with impaired gonadal function including Klinefelter syndrome.

The FSH receptor (FSHR) has been found to be expressed in human bone cells and bone marrow-adipocytes, and highly-debated mouse studies have suggested extra-gonadal effects of gonadotropins on glucose, adipocyte and bone homeostasis. These putative effects could be direct or indirectly mediated by endocrine factors released from bone-cells or adipocytes. Here, we investigated whether gonadotropins are linked with glucose- and lipid-metabolism in hypergonadotropic men. Single centre, cross-sectional study of 307 men with idiopathic infertility and 28 men with Klinefelter syndrome (KS). associations between serum LH and FSH with soluble-RANKL (sRANKL), osteoprotegerin (OPG), osteocalcin, fasting glucose and insulin, sex steroids, and body composition. Expression of FSHR was studied in human-derived adipocyte-cell-models (hMADS, TERT-hWA) and FSH stimulation of RANKL expression and secretion in hMADS in vitro. Serum FSH was not directly linked with glucose- and lipid-metabolism. However, FSH was inversely associated with sRANKL in both infertile men and KS men (p = .023 and p = .012). Infertile men with elevated FSH (>11 U/L) had significantly lower sRANKL (p = .015). sRANKL was positively associated with fat percentage, fasting insulin, and glucose (all p < .05). Men with prediabetes had higher sRANKL (p = .021), but lower testosterone (p < .0001) and Inhibin B (p = .005). The FSHR was expressed in the investigated human derived adipocytes, and 3-6 h treatment with FSH markedly increased RANKL release (p < .05). KS and infertile men with prediabetes have low Inhibin B, and testosterone but elevated RANKL compared with non-prediabetic men despite comparable levels of serum gonadotropins. Serum FSH and sRANKL was inversely associated in both infertile and KS men, but the increased release of RANKL from FSH treated adipocytes suggest a direct effect of FSH on RANKL production in some tissues. Further studies are required to clarify whether FSH targets RANKL in the skeleton. ClinicalTrial_ID:NCT01304927.

Abstract 4147: Neuropeptide Y promotes osteolytic activity during bone invasion and metastasis in Ewing sarcoma

Abstract Ewing sarcoma (ES) is a pediatric malignancy affecting bones and soft tissues. The presence of metastases is associated with poor prognosis, particularly in patients with dissemination to the bone. ES cells produce and secrete high levels of neuropeptide Y (NPY), a 36-amino acid neurotransmitter normally released from peripheral sympathetic neurons, and express its receptors. Our previous studies in an ES xenograft model demonstrated that the severity of bone destruction in primary tumors and incidence of osseous metastases positively correlate with levels of NPY release and are significantly decreased with NPY shRNA. Thus, the goal of our study was to determine the mechanism of this NPY-induced osteolytic effect. Since tumor bone invasion and metastasis is associated with changes in osteolytic and osteogenic activity within bone, we assessed osteoblast and osteoclast content in bones adjacent to the ES xenografts. No significant difference in osteoblast number was observed between bones within ES xenografts with high and low NPY levels. However, TRAP assay showed that ES tumors secreting high NPY levels had significantly increased osteoclast density on the border of the bone-tumor interface, as compared to tumor tissues with minimal NPY secretion or treated with NPY shRNA. To identify the mechanisms underlying these osteolytic actions of NPY, we tested its effect on osteoclast recruitment and differentiation. Transwell migration assay showed that osteoclast precursors, RAW 264.7 murine macrophages, were recruited at significantly higher levels to the conditioned media from ES cells with high NPY release, as compared to those cells exposed to conditioned media from NPY-low ES cells. Similarly, ES-conditioned media with high NPY content had a higher ability to stimulate differentiation of RAW 264.7 cells into osteoclasts than those obtained from NPY-low ES cells. Both of these effects were exacerbated by hypoxic conditions, known to upregulate NPY and its Y5 receptors (Y5R) in ES cells. Blocking the expression of NPY and Y5R in ES cells with shRNA inhibited macrophage recruitment and osteoclastogenesis, and decreased RANKL content in conditioned media from NPY-high ES cells. Altogether, these data suggest that the NPY/Y5R autocrine loop stimulates the release of RANKL from ES cells, promoting macrophage recruitment and consequent osteoclast differentiation. Therefore, increased NPY secretion could have a significant impact on shifting bone homeostasis to promote osteolysis, bone invasion, and osseous metastasis. Further studies are needed to identify other factors released by tumor cells secreting high NPY levels and to test Y5R antagonists as a potential therapeutic option to inhibit these processes. Importantly, aside from ES, the above findings may be relevant to other NPY-rich tumors metastasizing to the bone, such as neuroblastoma. Citation Format: Richard Garner, Emily Hong, Shiya Zhu, Mina Adnani, Sung-Hyeok Hong, Susana Galli, Joanna Kitlinska. Neuropeptide Y promotes osteolytic activity during bone invasion and metastasis in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4147.

Mechanisms of bone destruction in multiple myeloma.

Osteolytic bone disease is a frequent complication of multiple myeloma, resulting in skeletal complications that are a significant cause of morbidity and mortality. It is the result of an increased activity of osteoclasts, which is not followed by reactive bone formation by osteoblasts. Recent studies have revealed novel molecules and pathways that are implicated in osteoclast activation and osteoblast inhibition. Among them, the most important include the receptor activator of nuclear factor-kappa B ligand/osteoprotegerin pathway, the macrophage inflammatory proteins and the activin-A that play a crucial role in osteoclast stimulation in myeloma, while the wingless-type (Wnt) signalling inhibitors (sclerostin and dickkopf-1) along with the growth factor independence-1 are considered the most important factors for the osteoblast dysfunction of myeloma patients. Finally, the role of osteocytes, which is the key cell for normal bone remodelling, has also revealed during the last years through their interaction with myeloma cells that leads to their apoptosis and the release of RANKL and sclerostin maintaining bone loss in these patients. This review focuses on the latest available data for the mechanisms of bone destruction in multiple myeloma.

RANKL release from self-assembling nanofiber hydrogels for inducing osteoclastogenesis in vitro.

To develop a nanofiber hydrogel (NF-hydrogel) for sustained and controlled release of the recombinant receptor activator of NF-kB ligand; (RANKL) and to characterize the release kinetics and bioactivity of the released RANKL. Various concentrations of fluorescently-labelled RANKL protein were added to NF-hydrogels, composed of Acetyl-(Arg-Ala-Asp-Ala)4-CONH2 [(RADA)4] of different concentrations, to investigate the resulting in vitro release rates. The nano-structures of NF-hydrogel, with and without RANKL, were determined using atomic force microscopy (AFM). Released RANKL was further analyzed for changes in secondary and tertiary structure using CD spectroscopy and fluorescent emission spectroscopy, respectively. Bioactivity of released RANKL protein was determined using NFATc1 gene expression and tartrate resistant acid phosphatase (TRAP) activity of osteoclast cells as biomarkers. NF-hydrogel concentration dependent sustained release of RANKL protein was measured at concentrations between 0.5 and 2%(w/v). NF-hydrogel at 2%(w/v) concentration exhibited a sustained and slow-release of RANKL protein up to 48h. Secondary and tertiary structure analyses confirmed no changes to the RANKL protein released from NF-hydrogel in comparison to native RANKL. The results of NFATc1 gene mRNA expression and TRAP activities of osteoclast, showed that the release process did not affect the bioactivity of released RANKL. This novel study is the first of its kind to attempt in vitro characterization of NF-hydrogel based delivery of RANKL protein to induce osteoclastogenesis. We have shown the self-assembling NF-hydrogel peptide system is amenable to the sustained and controlled release of RANKL locally; that could in turn increase local concentration of RANKL to induce osteoclastogenesis, for application to the controlled mobilization of tooth movement in orthodontic procedures. Orthodontic tooth movement (OTM) occurs through controlled application of light forces to teeth, facilitating the required changes in the surrounding alveolar bone through the process of bone remodelling. The RANKL system regulates alveolar bone remodelling and controls root resorption during OTM. The use of exogenous RANKL to accelerate OTM has not been attempted to date because large quantities of RANKL for systemic therapy may subsequently cause serious systemic loss of skeletal bone. The controlled and sustained local release of RANKL from a carrier matrix could maximize its therapeutic benefit whilst minimizing systemic side effects. In this study a NF-hydrogel was used for sustained and controlled release of RANKL and the release kinetics and biofunctionality of the released RANKL was characterized. Our results provide fundamental insight for further investigating the role of RANKL NF-hydrogel release systems for inducing osteoclastogenesis in vivo.

The Role of IL-17 and TH17 Cells in the Bone Catabolic Activity of PTH.

Osteoimmunology is field of research dedicated to the study of the interactions between the immune system and bone. Among the cells of the immune system that regulate the skeleton in health and disease are T lymphocytes, T cells secrete inflammatory/osteoclastogenic cytokines such as RANKL, TNF, and IL-17, as well as factors that stimulate bone formation, including Wnt ligands. In addition, T cells regulate the differentiation and life span of stromal cells via CD40L and other costimulatory molecules expressed on their surface. Consensus exists that parathyroid hormone (PTH) induces bone loss by increasing the production of RANKL by osteocytes and osteoblast. However, new evidence suggests that PTH expands Th17 cells and increases IL-17 levels in mice and humans. Studies in the mouse of further shown that Th17 cell produced IL-17 acts as an “upstream cytokine” that increases the sensitivity of osteoblasts and osteocytes to PTH. As a result, PTH stimulates osteocytic and osteoblastic release of RANKL. Therefore, PTH cause bone loss only in the presence of IL-17 signaling. This article reviews the evidence that the effects of PTH are mediated not only by osteoblasts and osteocytes, but also T cells and IL-17.

Glutathione, N-acetylcysteine and lipoic acid down-regulate starvation-induced apoptosis, RANKL/OPG ratio and sclerostin in osteocytes: involvement of JNK and ERK1/2 signalling.

Osteocyte apoptosis due to microdamage and/or oxidative stress is related to increased local bone turnover and resorption observed in various bone diseases. Previous data on osteoblasts and osteoclasts have linked reactive oxygen species and antioxidants to bone remodelling. This study performs a comprehensive analysis on the effect of antioxidants such as glutathione (GSH), N-acetylcysteine and lipoic acid (LA) on starvation-induced osteocyte apoptosis and on cytokines involved in bone remodelling such as the receptor activator kB ligand (RANKL), osteoprotegerin (OPG) and sclerostin. For this study, apoptosis was induced by serum starvation in a murine osteocyte-like cell line MLO-Y4; this condition mimics in part osteocyte apoptosis due to microdamage. The results show that starvation-induced apoptosis and expression of RANKL, OPG and sclerostin are redox regulated processes. All antioxidants are able to inhibit the apoptosis due to starvation. They down-regulate the expression and the release of RANKL, the expression of sclerostin and RANKL/OPG ratio, whereas they only in part up-regulate OPG expression. Antioxidants mediate their effect on starvation-induced apoptosis by JNK signalling and on cytokine expression by both JNK and ERK1/2 activities. This study shows the possible involvement of biological antioxidants such as GSH and LA on redox regulated mechanisms related to apoptosis and expression of cytokines involved in bone remodelling. Moreover, it suggests that both JNK and ERK1/2 may be useful biological targets for drugs affecting bone diseases associated with increased oxidative stress.

Incorporation of RANKL promotes osteoclast formation and osteoclast activity on β-TCP ceramics

β-Tricalcium phosphate (β-TCP) ceramics are approved for the repair of osseous defects. In large defects, however, the substitution of the material by authentic bone is inadequate to provide sufficient long-term mechanical stability. We aimed to develop composites of β-TCP ceramics and receptor activator of nuclear factor κ-B ligand (RANKL) to enhance the formation of osteoclasts and promote cell mediated calcium phosphate resorption. RANKL was adsorbed superficially onto β-TCP ceramics or incorporated into a crystalline layer of calcium phosphate by the use of a co-precipitation technique. Murine osteoclast precursors were seeded onto the ceramics. After 15days, the formation of osteoclasts was quantified cytologically and colorimetrically with tartrate-resistant acidic phosphatase (TRAP) staining and TRAP activity measurements, respectively. Additionally, the expression of transcripts encoding the osteoclast gene products cathepsin K, calcitonin receptor, and of the sodium/hydrogen exchanger NHA2 were quantified by real-time PCR. The activity of newly formed osteoclasts was evaluated by means of a calcium phosphate resorption assay. Superficially adsorbed RANKL did not induce the formation of osteoclasts on β-TCP ceramics. When co-precipitated onto β-TCP ceramics RANKL supported the formation of mature osteoclasts. The development of osteoclast lineage cells was further confirmed by the increased expression of cathepsin K, calcitonin receptor, and NHA2. Incorporated RANKL stimulated the cells to resorb crystalline calcium phosphate. Our in vitro study shows that RANKL incorporated into β-TCP ceramics induces the formation of active, resorbing osteoclasts on the material surface. Once formed, osteoclasts mediate the release of RANKL thereby perpetuating their differentiation and activation. In vivo, the stimulation of osteoclast-mediated resorption may contribute to a coordinated sequence of material resorption and bone formation. Further in vivo studies are needed to confirm the current in vitro findings.

RANKL subcellular trafficking and regulatory mechanisms in osteocytes

The receptor activator of the NF-κB ligand (RANKL) is the central player in the regulation of osteoclastogenesis, and the quantity of RANKL presented to osteoclast precursors is an important factor determining the magnitude of osteoclast formation. Because osteoblastic cells are thought to be a major source of RANKL, the regulatory mechanisms of RANKL subcellular trafficking have been studied in osteoblastic cells. However, recent reports showed that osteocytes are a major source of RANKL presentation to osteoclast precursors, prompting a need to reinvestigate RANKL subcellular trafficking in osteocytes. Investigation of molecular mechanisms in detail needs well-designed in vitro experimental systems. Thus, we developed a novel co-culture system of osteoclast precursors and osteocytes embedded in collagen gel. Experiments using this model revealed that osteocytic RANKL is provided as a membrane-bound form to osteoclast precursors through osteocyte dendritic processes and that the contribution of soluble RANKL to the osteoclastogenesis supported by osteocytes is minor. Moreover, the regulation of RANKL subcellular trafficking, such as OPG-mediated transport of newly synthesized RANKL molecules to lysosomal storage compartments, and the release of RANKL to the cell surface upon stimulation with RANK are confirmed to be functional in osteocytes. These results provide a novel understanding of the regulation of osteoclastogenesis.

RANKL Expression, Function, and Therapeutic Targeting in Multiple Myeloma and Chronic Lymphocytic Leukemia

Bone destruction is a prominent feature of multiple myeloma, but conflicting data exist on the expression and pathophysiologic involvement of the bone remodeling ligand RANKL in this disease and the potential therapeutic benefits of its targeted inhibition. Here, we show that RANKL is expressed by primary multiple myeloma and chronic lymphocytic leukemia (CLL) cells, whereas release of soluble RANKL was observed exclusively with multiple myeloma cells and was strongly influenced by posttranscriptional/posttranslational regulation. Signaling via RANKL into multiple myeloma and CLL cells induced release of cytokines involved in disease pathophysiology. Both the effects of RANKL on osteoclastogenesis and cytokine production by malignant cells could be blocked by disruption of RANK-RANKL interaction with denosumab. As we aimed to combine neutralization of RANKL with induction of antibody-dependent cellular cytotoxicity of natural killer (NK) cells against RANKL-expressing malignant cells and as denosumab does not stimulate NK reactivity, we generated RANK-Fc fusion proteins with modified Fc moieties. The latter displayed similar capacity compared with denosumab to neutralize the effects of RANKL on osteoclastogenesis in vitro, but also potently stimulated NK cell reactivity against primary RANKL-expressing malignant B cells, which was dependent on their engineered affinity to CD16. Our findings introduce Fc-optimized RANK-Ig fusion proteins as attractive tools to neutralize the detrimental function of RANKL while at the same time potently stimulating NK cell antitumor immunity.

Local effect of IL‐4 delivery on polyethylene particle induced osteolysis in the murine calvarium

Wear particles generated with use of total joint replacements incite a chronic macrophage-mediated inflammatory reaction, which leads to implant failure. Macrophage activation may be polarized into two states, with an M1 proinflammatory state dominating an alternatively activated M2 anti-inflammatory state. We hypothesized that IL-4, an activator of M2 macrophages, could modulate polyethylene (PE) particle-induced osteolysis in an experimental murine model. Four animal groups included (a) calvarial saline injection with harvest at 14 days (b) single calvarial injection of PE particles subcutaneously (SC) without IL-4 (c) PE particles placed as in (b), then IL-4 given SC for 14 consecutive days and (d) PE particles as in (b) then IL-4 beginning 7 days after particle injection for 7 days. The calvarial bone volume to total tissue volume was measured using microCT and histomorphometry. Calvaria were cultured for 24 h to assess release of RANKL, OPG, TNF-α, and IL-1ra and isolation and identification of M1 and M2 specific proteins. MicroCT and histomorphometric analysis showed that bone loss was significantly decreased following IL-4 administration to PE treated calvaria for both 7 and 14 days. Western blot analysis showed an increased M1/M2 ratio in the PE treated calvaria, which decreased with addition of IL-4. Cytokine analysis showed that the RANKL/OPG ratio and TNF-α/IL-1ra ratio decreased in PE-treated calvaria following IL-4 addition for 14 days. IL-4 delivery mitigated PE particle-induced osteolysis through macrophage polarization. Modulation of macrophage polarization is a potential treatment strategy for wear particle induced periprosthetic osteolysis.

Relation of interleukin-6 in rheumatoid arthritis patients to systemic bone loss and structural bone damage

IL-6 plays a key role in local and systemic manifestation of RA. IL-6 is not only a pro-inflammatory cytokine, but also interacts in complex ways with the cells involved in bone remodeling. In RA, IL-6 may indirectly promote osteoclastogenesis by increasing the release of RANK-L by osteoblasts, and it diminishes the proliferation of osteoblasts at late differentiation stages. The aims of this work were to evaluate the level of serum IL-6 and to correlate it with the activity, severity, early development of osteoporosis, and early structural bone damage in RA patients. The following parameters were investigated in 40 RA patients and 20 healthy controls: IL-6 level, BMI, ESR, CRP, CBCs, serum ionized calcium, blood urea, serum creatinine, AST, ALT, anti-CCP, and RF. Bone mineral density was measured by dual-energy X-ray absorptiometry at lumbar spines and femoral neck. Drug history was taken stressing on steroid therapy. Data were processed and analyzed using computer-based program. IL-6 was significantly positively correlated with HAQ1, PTGA, grade of pain, ESR, platelet count, blood urea, AST level, and anti-CCP level; IL-6 showed an inverse significant correlation with T-score. IL-6 was positively correlated with TGC, DAS-28 score, and RF level. No correlation was found between T-score and morning stiffness duration, BMI, CRP, RBC, serum creatinine, and ALT. There was an inverse significant correlation between T-score and HAQ1, SJC, pain grade, DAS-28 score, PTGA, ESR, RF, anti-CCP, and IL-6. Patients with RA on steroid therapy had significantly higher TJC, SJC, and DAS-28 score, anti-CCP, and IL-6 than patients with RA not on steroid therapy. Patients with RA on steroid therapy had significantly lower T-score and lower serum ionized calcium than patients with RA not on steroid therapy. IL-6 has an important role in increasing osteoclastic activity and subsequent bone resorption in the patients with RA. Blocking IL-6 by using IL-6 inhibitors and anti-RANK-L therapy may be effective in inhibiting the inflammatory process and preventing the bone complications of RA disease.

Effect of heat stress on the expression levels of receptor activator of NF‐κB ligand and osteoprotegerin in human periodontal ligament cells

To investigate the expression levels of the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) in human periodontal ligament fibroblasts (PDLs) when stimulated with heat. Periodontal ligament fibroblasts were subjected to various temperature increases for 5 min and then maintained at 37 °C. After that, cell viability was determined using an MTT assay. The expression levels of RANKL and OPG were investigated using real-time RT-PCR and ELISA. As a control, the cells were cultured at 37 °C. Data were analysed using one-way anova at a significant level of P = 0.05. Results Cell viability was reduced significantly in the heated groups (P < 0.05). Heat stress downregulated the mRNA expression levels of RANKL and OPG (P < 0.05). When the cells were heated at 39 °C, the protein release of OPG was increased (P < 0.05). There were no significant differences between the heated groups and the control in the release of soluble RANKL (P > 0.05). The relative RANKL/OPG expression ratios were decreased at 39, 43 and 50 °C (P < 0.05), but increased at 47 °C (P < 0.05). Heat influenced the balance between RANKL and OPG in PDLs. Low heat downregulated their relative ratio, whilst high heat upregulated it.

Role for interleukin-6 in structural joint damage and systemic bone loss in rheumatoid arthritis

Interleukin-6 (IL-6) plays a key role in the local and systemic manifestations of rheumatoid arthritis (RA). IL-6 is not only a proinflammatory cytokine, but also interacts in complex ways with the cells involved in bone remodeling. In RA, IL-6 may indirectly promote osteoclastogenesis by increasing the release of RANK-L by osteoblasts and synovial cells. However, IL-6 inhibits osteoclastogenesis in vitro, via a direct mechanism. The effects of IL-6 on osteoblasts may vary with the cell differentiation stage: thus, IL-6 may promote the differentiation of pre-osteoblasts to mature osteoblasts while also diminishing the proliferation of osteoblasts at late differentiation stages. Thus, the effects of IL-6 on bone remodeling are complex and may occur in opposite directions depending on the model or experimental conditions. Nevertheless, results from studies in animal models and humans support a negative effect of IL-6 on bone. Thus, in patients with RA, blocking IL-6 may be effective both in diminishing the inflammatory manifestations and in preventing the bone complications of the disease.

Microdamage detection and repair in bone: Fracture mechanics, histology, cell biology

Bone is an elementary component in the human skeleton. It protects vital organs, regulates calcium levels and allows mobility. As a result of daily activities, bones are cyclically strained causing microdamage. This damage, in the form of numerous microcracks, can cause bones to fracture and therefore poses a threat to mechanical integrity. Bone is able to repair the microcracks through a process called remodelling which is tightly regulated by bone forming and resorbing cells. However, the manner by which microcracks are detected, and repair initiated, has not been elucidated until now. Here we show that microcrack accumulation causes damage to the network of cellular processes, resulting in the release of RANKL which stimulates the differentiation of cells specialising in repair.

Osteoprotegerin Reduces the Serum Level of Receptor Activator of NF-κB Ligand Derived from Osteoblasts

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of NF-kappaB ligand (RANKL). We previously reported that OPG deficiency elevated the circulating level of RANKL in mice. Using OPG(-/-) mice, we investigated whether OPG is involved in the shedding of RANKL by cells expressing RANKL. Osteoblasts and activated T cells in culture released a large amount of RANKL in the absence of OPG. OPG or a soluble form of receptor activator of NF-kappaB (the receptor of RANKL) suppressed the release of RANKL from those cells. OPG- and T cell-double-deficient mice showed an elevated serum RANKL level equivalent to that of OPG(-/-) mice, indicating that circulating RANKL is mainly derived from bone. The serum level of RANKL in OPG(-/-) mice was increased by ovariectomy or administration of 1alpha,25-dihydroxyvitamin D(3). Expression of RANKL mRNA in bone, but not thymus or spleen, was increased in wild-type and OPG(-/-) mice by 1alpha,25-dihydroxyvitamin D(3). These results suggest that OPG suppresses the shedding of RANKL from osteoblasts and that the serum RANKL in OPG(-/-) mice exactly reflects the state of bone resorption.

CCL20 chemokine induces both osteoblast proliferation and osteoclast differentiation: Increased levels of CCL20 are expressed in subchondral bone tissue of rheumatoid arthritis patients

We evaluated the role of CCL20 (MIP-3alpha) chemokine in cells directly involved in the remodeling of bone tissue (osteoblasts and osteoclasts) and we confirmed its expression in the subchondral bone tissue of rheumatoid arthritis (RA) patients. The expression of CCL20 and of its receptor CCR6 was evaluated in osteoblasts isolated from bone tissue of post-traumatic (PT) patients. Functional tests were performed to evaluate osteoblast proliferation and matrix protein modulation. Immunohistochemical analysis for CCR6, CCL20, and RANKL was performed on bone samples from RA patients. The role of CCL20 was then analyzed in osteoclast differentiation. We found that in basal conditions CCR6, but not its ligand CCL20, was highly expressed by osteoblasts. Functional analysis on osteoblasts showed that CCL20 significantly increased cellular proliferation but did not affect matrix protein expression. Pro-inflammatory cytokines significantly induced the release of CCL20 and RANKL by human osteoblasts but did not modulate CCR6 expression. Increased expression of CCR6, CCL20, and RANKL was confirmed in RA subchondral bone tissue biopsies. We demonstrated that CCL20 was also an earlier inducer of osteoclast differentiation by increasing the number of pre-osteoclasts, thus favoring cell fusion and MMP-9 release. Our results add new insight to the important role of the CCL20/CCR6, RANKL system in the bone tissue of RA. The contemporary action of CCL20 on osteoblasts and osteoclasts involved in the maintenance of bone tissue homeostasis demonstrates the important role of this compartment in the evolution of RA, by showing a clear uncoupling between new bone formation and bone resorption.