Receptor Activator Of Nuclear Factor kappa-B Expression Research Articles

Magnesium Lithospermate B Protects against Lipopolysaccharide-Induced Bone Loss by Inhibiting RANKL/RANK Pathway.

Lipopolysaccharide (LPS) can induce bone loss by stimulating bone resorption. Natural compounds have great potential for the treatment of osteolytic bone diseases. Magnesium lithospermate B (MLB) plays an important role in protecting against oxidative damage and also has potential anti-inflammatory pharmacological properties. However, its role in LPS-induced bone loss is still unknown. In the present study, we observed the effects of MLB on LPS-induced bone damage and investigated the possible mechanisms. The bone loss models were established by LPS administration in male Sprague–Dawley rats. MLB (200 mg/kg body weight) was given by subcutaneous injection. MicroCT analysis, biomarker assay, histological examination and immunohistochemical staining were performed at the 8th weeks. In addition, RAW264.7 cells were treated with LPS in the presence or absence of MLB. The osteoclast formation, resorption activity and differentiation-related genes [(receptor activator of nuclear factor kappa-B (RANK), Traf6, Fra-1, and c-src)] expression were evaluated. LPS induced bone loss shown as the decrease in bone volume fraction and trabecular number, and increase in trabecular separation. LPS also markedly enhanced the osteoclast formation and resorption activity compared with the control. MLB significantly abolished the LPS-induced bone microstructure damage (p < 0.05) and osteoclast formation. MLB also inhibited the increases of serum tartrate-resistant acid phosphatase 5b, RANK ligand (RANKL) and TNF-α level enhanced by LPS (p < 0.05). Immunohistochemical staining indicated that MLB attenuated the high expression of RANKL and RANK stimulated by LPS. In addition, MLB significantly abolished the LPS-enhanced osteoclast formation, resorption activity, RANK, Traf6, Fra-1, and c-src expression in vitro. Our data demonstrate that MLB can suppress LPS-induced bone loss via inhibiting RANKL/RANK related osteoclast formation.

Prognostic value of receptor activator of nuclear factor kappa-B (RANK) marker in patients with breast cancer

Despite notable progress made in studying breast cancer (BC), the mechanisms of metastases, in view of the classification into molecular subtypes, in patients with BC remain to be fully uninvestigated, in the presence of a good prognosis in particular. To study novel diagnostic and predictive markers in a new way presents current problems in the pathology of BC. This investigation deals with the expression of osteoprotegerin (OPG) in the tumor cells of patients with BC. It enrolled 83 patients with locally advanced BC (T2–4N0–3M0) who had been treated in 2003 to 2010. The inclusion criterion was a histologically verified diagnosis of invasive BC. To study the level of OPG, the investigators conducted an immunohistochemical study of biopsy sections according to the standard protocol. The mean expression of receptor activator of nuclear factor kappa-B (RANK) in the BC cells was 18.7 %; its median was 5 % (range, 0–90 %). The patients were divided into 2 groups according to the level of RANK expression: 1) high (higher than the median); 2) low (lower than the median). The high RANK group included 39 patients; the low RANK group comprised 44 patients. Analysis of the clinical and pathological characteristics of BC patients with regard RANK expression did not show any statistically significant differences in the presence or absence of affected regional lymph nodes, T category, and Ki-67 index. The analysis of clinical and pathomorphological and immunohistochemical characteristics in patients with breast cancer, taking into consideration RANK expression level, did not show any statistically significant differences with respect to presence or absence of affected regional lymph nodes, age, T category and Ki-67 index (р &gt; 0.05). However, it revealed the following pattern: the high expression of RANK was more common in patients positive for estrogen and progesterone receptors than in those for negative receptors (p = 0.04).

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.

Expression profile of osteoprotegerin, RANK and RANKL genes in the femoral head of patients with avascular necrosis

IntroductionFemoral head avascular necrosis (AVN) is a recalcitrant disease of the hip that leads to joint destruction. Osteoprotegerin (OPG), Receptor Activator of Nuclear Factor kappa-B (RANK) and RANK ligand (RANKL) regulate the balance between osteoclasts–osteoblasts. The expression of these genes affects the maturation and function of osteoblasts–osteoclasts and bone remodeling. In this study, we investigated the molecular pathways leading to AVN by studying the expression profile of OPG, RANK and RANKL genes. Material and methodsQuantitative Real Time-PCR was performed for evaluation of OPG, RANK and RANKL expression. Analysis was based on parallel evaluation of mRNA and protein levels in normal/necrotic sites of 42 osteonecrotic femoral heads (FHs). OPG and RANKL protein levels were estimated by western blotting. ResultsThe OPG mRNA levels were higher (insignificantly) in the necrotic than the normal site (p>0.05). Although the expression of RANK and RANKL was significantly lower than OPG in both sites, RANK and RANKL mRNA levels were higher in the necrotic part than the normal (p<0.05). Protein levels of OPG and RANKL showed no remarkable divergence. ConclusionsOur results indicate that differential expression mechanisms for OPG, RANK and RANKL that could play an important role in the progress of bone remodeling in the necrotic area, disturbing bone homeostasis. This finding may have an effect on the resulting bone destruction and the subsequent collapse of the hip joint.

Expression of Toll‐like receptors 2 and 4 and the OPG–RANKL–RANK system in inflammatory external root resorption and external cervical resorption

To investigate and compare the expression of the pathogen recognition receptors Toll-like receptor (TLR) 2 and TLR4, and the hard tissue resorption triad osteoprotegerin (OPG)-receptor activator of nuclear factor kappa-B ligand (RANKL)-receptor activator of nuclear factor kappa-B (RANK) in external inflammatory root resorption of endodontic origin (ER) and external cervical root resorption (ECR) by immunohistochemistry. Formalin-fixed, paraffin-embedded archival specimens collected from teeth that were diagnosed clinically, radiographically and histopathologically with either ER (n = 9) or ECR (n = 9) were processed for immunohistochemistry to investigate and compare levels of TLR2, TLR4, OPG, RANKL, RANK, CD3, CD19 and CD83 expression. The histological features were evaluated via haematoxylin and eosin stain. Taylor's modification of the Brown and Brenn Gram stain was used for examining the presence and distribution of bacteria. All stained slides were digitally photographed and qualitatively analysed, and F test and unpaired Student's t-test were used for statistical analysis. Both ER and ECR showed similar immuno-histopathology characteristics of a fibrovascular connective tissue with varying degrees of inflammatory infiltrate consisting of T and B lymphocytes, dendritic cells, polymorphonuclear lymphocytes and plasma cells. Colonies of bacteria were identified in the majority of lesions, and this correlated with the cellular expression of TLR2 and TLR4 in all lesions. Similarly, all lesions showed a significantly higher (P < 0.05) level of cells expressing RANKL than OPG, indicating hard tissue resorption processes where active in the lesions. The immunohistopathology patterns of ECR samples were consistent with the bacteria-driven ER specimens, suggesting bacteria-induced inflammation may be involved in ECR.

Osteoprotegerin (OPG) and Related Proteins (RANK, RANKL and TRAIL) in Thyroid Disease

Osteoprotegerin (OPG), receptor activator of nuclear factor kappaB (RANK), RANK ligand (RANKL), and TNF related apoptosis inducing ligand (TRAIL) are the key proteins in the development of bone metastases. Osteoprotegerin improves tumor cell survival and inhibits TRAIL induced apoptosis of cancer cell lines. It also binds to RANKL and inhibits its interaction with RANK (cell surface receptor), which influences osteoclast formation and function. The aim of the present study was to characterize the expression of OPG, RANK, RANKL, and TRAIL in benign and malignant thyroid tissue and thyroid cell lines. Archived thyroid tissue from 79 patients (60 differentiated thyroid cancers and 19 benign thyroids) was stained for OPG, RANK, RANKL, and TRAIL by immunohistochemistry. Staining was assessed semiquantitatively and correlated with pathology. Western blots were performed on three human thyroid cell lines: Nthy-ori 3-1 (thyroid follicular epithelium), FTC-133 (follicular thyroid carcinoma), and K1E7 (subclone of papillary thyroid carcinoma). Osteoprotegerin and RANKL staining was cytoplasmic; RANK staining was nuclear; and TRAIL staining was predominantly cytoplasmic. All four proteins were expressed in benign and malignant tissue. There was significant difference in RANK expression between malignant and benign tissue (8 vs. 84%, respectively; Fisher's exact test; P < 0.001). RANKL expression was significantly increased in malignant tissue (Fisher's exact test; P = 0.04). Western blotting showed OPG expression in all cell lines and TRAIL in none. RANK and RANKL were not detected in papillary and follicular cell lines, respectively. Nuclear expression of RANK protein in thyroid tissue is paradoxical; it could be due to nuclear migration after ligand binding. Suppression of RANK and increased expression of RANKL in malignant tissue warrant further investigation.

Examination of the mechanisms of osteolysis in patients with metastatic breast cancer

The benefits of bisphosphonates (BPs) in reducing skeletal-related events (SREs) in patients with bone metastases has mainly been attributed to their potent osteoclast inhibiting effect. However, despite the use of modern systemic anticancer therapy including potent BPs, many patients with bone metastases continue to have SREs. An improved understanding of the fundamental mechanisms of bone destruction allows for further development of appropriate targeted treatments. In this study, archival paraffin-embedded bone metastases specimens from patients with metastatic breast cancer were examined for the presence of osteoclasts, expression of the receptor activator of nuclear factor kappaB (RANK), RANK Ligand (RANKL), osteoprotegerin (OPG) and vascular endothelial growth factor (VEGF). Histological specimens were available for primary breast cancer, lymph node metastases, normal breast and normal bone tissues for comparison. Bone metastasis specimens were available for 20 BP naïve patients and two BP-treated patients. Osteoclasts were significantly increased in the bone metastases of the BP naïve group compared to normal bone. No osteoclasts were detected in the BP-treated group. RANKL was predominantly expressed in osteoblasts and in the stromal elements of metastatic tissue. Conversely, RANK was present in osteoclasts of bone metastases and normal bone, as well as in tumor cells of metastatic lymph nodes and bone metastases. VEGF was strongly expressed in the control bone and bone metastases regardless of BP treatment. In summary, osteoclasts may not be the singular obligatory factor for osteolysis in bone metastases. An increased expression of RANKL in stromal tissue surrounding bone metastases, RANK in osteoclasts and VEGF may serve as future targeted therapies possibly in conjunction with bisphosphonates. The mechanisms for osteoclast expression and the expression of RANKL, RANK, OPG and VEGF merit further prospective analysis, particularly in the context of BP treatment and progressive bone metastases.

Expression of receptor activator of nuclear factor -κB ligand, receptor activator of nuclear factor -κB, and osteoprotegerin, following low-level laser treatment on deproteinized bovine bone graft in rats

The aim of this study was to investigate by immunohistochemistry the effects of low-level laser (LLL) irradiation on the expression of the receptor activator of nuclear factor -kappaB ligand (RANKL), osteoprotegerin (OPG), and the receptor activator of nuclear factor -kappaB (RANK) in deproteinized bovine bone grafts in rats. Twenty-four male Sprague-Dawley rats aged 15 weeks were allocated to either an experimental group that underwent LLL irradiation during bone healing at the bone graft sites of the rats' calvarial bone defects or a control group. In the experimental group, gallium-aluminum-arsenide (Ga-Al-As) diode LLL (wavelength 808 nm; output 96 mW) was used to irradiate three areas on and around bone defects. The radiation was administered by the contact method for 10 s at 8.3 J/cm(2), once a day for 7 days. The total dose over the complete schedule was 40.32 J. The animals were killed on days 7, 14 or 21. The results of immunohistochemical analysis showed that the expression of RANKL (P = 0.199), OPG (P = 0.035), and RANK (P = 0.020) in the experimental group significantly increased from day 7, with a more even distribution than in the control group, and that this difference prevailed until the end of the experiment. Bone density of the experimental group after trichrome staining was also higher than in the control group. These results suggest that LLL irradiation facilitates bone metabolism during bone healing at the sites of deproteinized bovine bone grafts in rats.

Minodronic acid influences receptor activator of nuclear factor kB ligand expression and suppresses bone resorption by osteoclasts in rats with collagen-induced arthritis

We investigated the inhibitory mechanism of bone resorption by minodronic acid in collagen-induced arthritis (CIA) in rats. Four groups of female Sprague-Dawley rats, aged 7 months, were studied: three groups of collagen-sensitized rats, including one placebo-administered group (CIA-P), and two minodronic acid-administered groups at 0.2 mg/kg/2 day (CIA-BIS) and 2.0 mg/kg/2 day (CIA-BIS10). These were studied with an additional untreated observation group (Cont group). Minodronic acid was administered orally a day after the initial sensitization. The femoral posteromedial condyle was analyzed histologically and immunohistologically 4 weeks after the initial sensitization. Western blotting was also performed to assess the receptor activator of nuclear factor kappaB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) expression of the knee joints. In CIA-P rats, many tartrate-resistant acid phosphatase (TRAP)-positive cells were found at the pannus-lining layer and the epiphyseal medulla. The bone-lining cells in the epiphyseal medulla and the cells in the pannus strongly expressed RANK and RANKL. In the minodronic acid-administered group, the number of TRAP-positive cells and the severity of arthritis were reduced. The reduction in the CIA-BIS10 group was significant compared with the CIA-P group (P < 0.05). Dosage-dependent reduction of RANK and RANKL expression was confirmed by immunohistology and Western blotting. With or without minodronic acid administration, no apoptotic cells were found in any groups using the TdT-mediated dUTP-biotinnick end labeling (TUNEL) method. The expression of OPG was not clear in all groups. These results demonstrated that minodronic acid inhibited the differentiation and the activation of osteoclasts not by inducing apoptosis but by inhibiting the RANKL-RANK system, and thereby suppressing bone resorption.

Effects of disease‐modifying antirheumatic drugs and antiinflammatory cytokines on human osteoclastogenesis through interaction with receptor activator of nuclear factor κB, osteoprotegerin, and receptor activator of nuclear factor κB ligand

To demonstrate the effects of disease-modifying antirheumatic drugs and antiinflammatory cytokines on human osteoclastogenesis through their effects on receptor activator of nuclear factor kappaB (RANK), osteoprotegerin (OPG), and RANK ligand (RANKL). Peripheral blood mononuclear cells (PBMCs) and rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were cocultured in the presence of macrophage colony-stimulating factor, 1,25-dihydroxyvitamin D(3), and various concentrations of methotrexate (MTX), sulfasalazine (SSZ), hydroxychloroquine (HCQ), anti-tumor necrosis factor alpha monoclonal antibody (infliximab), interleukin-4 (IL-4), and IL-10. Osteoclast formation was assayed by counting cells after staining for tartrate-resistant acid phosphatase. RANKL expression in RA FLS and RANK expression in PBMCs were assayed by Western blotting, reverse transcription-polymerase chain reaction (RT-PCR), and real-time PCR. OPG expression was measured by enzyme-linked immunosorbent assay, RT-PCR, and real-time PCR in cultures of RA FLS. MTX, SSZ, infliximab, and IL-4, but not IL-10 and HCQ, each inhibited osteoclast formation in a dose-dependent manner. We observed no evidence of synergistic inhibition of osteoclast formation by IL-4 and IL-10. High doses of infliximab suppressed the expression of RANK in PBMCs. MTX, SSZ, infliximab, and IL-4 each inhibited the expression of RANKL in RA FLS in a dose-dependent manner, and also increased the secretion of OPG in RA FLS supernatants. MTX, SSZ, infliximab, and IL-4 inhibit human osteoclastogenesis by modulating the interaction of RANKL, RANK, and OPG. These results are indicative of the underlying mechanisms of the antiresorptive effects of these 4 agents.

In situ expression of RANKL, RANK, osteoprotegerin and cytokines in osteoclasts of rat periodontal tissue.

This study examined the in situ expression of receptor activator of nuclear factor-kappaB ligand (RANKL), receptor activator of nuclear factor-kappaB (RANK), osteoprotegerin, interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) in the osteoclasts of rat periodontal tissue. In periodontal disease, osteoclasts cause resorption of the alveolar bone. The function of osteoclasts is regulated by interaction with periodontal ligament cells (PDLs). Furthermore, various kinds of molecules such as RANKL, RANK, osteoprotegerin, IL-1beta and TNFalpha are known to be related to the osteoclasts differentiation and function. It is therefore important to observe the expression of RANKL, RANK, osteoprotegerin and cytokines in osteoclasts and PDLs. Four-week-old Wistar rats were used. Tooth movement was performed by the Waldo method, and the pathological bone resorption was induced. The demineralized maxillae and mandiblae were embedded with paraffin. In situ hybridization was performed to detect RANKL, RANK, osteoprotegerin, IL-1beta, and TNFalpha mRNAs in osteoclasts and other cells using the specific RNA probes, respectively. Both RANKL and RANK were concomitantly expressed in some osteoclasts. RANKL was also positive in osteoblasts and PDLs. No IL-1beta- and TNFalpha-positive osteoclast was noted. The positive signals of osteoprotegerin were detected in almost all osteoblasts, PDLs and odontoblasts. No osteoprotegerin-positive osteoclasts were observed. The number and the distribution pattern of RANKL- and RANK-expressing osteoclasts changed when orthodontic excessive force was applied to periodontal tissue. In addition, IL-1beta and TNFalpha were shown to be expressed in osteoclasts under pathological status. These findings suggest that an autocrine mechanism of RANKL-RANK exists in osteoclast, which is heightened in the pathological conditions. Furthermore, the autocrine mechanism of IL-1beta and TNFalpha is also provided in osteoclast under pathological condition. These autocrine mechanisms therefore seem to regulate the osteoclast function in both physiological and pathological conditions.

Activin A stimulates IkappaB-alpha/NFkappaB and RANK expression for osteoclast differentiation, but not AKT survival pathway in osteoclast precursors.

Recent studies have reported that activin A enhances osteoclastogenesis in cultures of mouse bone marrow cells stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). However, the exact mechanisms by which activin A functions during osteoclastogenesis are not clear. RANKL stimulation of RANK/TRAF6 signaling increases nuclear factor-kappaB (NFkappaB) nuclear translocation and activates the Akt/PKB cell survival pathway. Here we report that activin A alone activates IkappaB-alpha, and stimulates nuclear translocation of NFkappaB and receptor activator of nuclear factor-kappaB (RANK) expression for osteoclastogenesis, but not Akt/PKB survival signal transduction including BAD and mammalian target of rapamycin (mTOR) for survival in osteoclast precursors in vitro. Activin A alone failed to activate Akt, BAD, and mTOR by immunoblotting, and it also failed to prevent apoptosis in osteoclast precursors. While activin A activated IkappaB-alpha and induced nuclear translocation of phosphorylated-NFkappaB, and it also enhanced RANK expression in osteoclast precursors. Moreover, activin A enhanced RANKL- and M-CSF-stimulated nuclear translocation of NFkappaB. Our data suggest that activin A enhances osteoclastogenesis treated with RANKL and M-CSF via stimulation of RANK, thereby increasing the RANKL stimulation. Activin A alone activated the NFkappaB pathway, but not survival in osteoclast precursors in vitro, but it is, thus, insufficient as a sole stimulus to osteoclastogenesis.

Increase in expression of receptor activator of nuclear factor kappaB at sites of bone erosion correlates with progression of inflammation in evolving collagen-induced arthritis.

The receptor activator of nuclear factor kappaB (RANK)/RANK ligand (RANKL) pathway is critical in osteoclastogenesis and bone resorption and has been implicated in the process of focal bone erosion in arthritis. This study was undertaken to identify in vivo the hitherto-unknown origin and localization of RANK-expressing osteoclast precursor cells at sites of bone erosion in arthritis. DBA-1 mice were immunized with bovine type II collagen/Freund's complete adjuvant and were given an intraperitoneal booster injection of type II collagen on day 21. Arthritis was monitored visually, and joint pathology was examined histologically. RANK and RANKL expression were analyzed using specific immunohistochemistry, and tartrate-resistant acid phosphatase (TRAP) staining was performed. In addition, TRAP and cathepsin K messenger RNA expression were analyzed by in situ hybridization. A marked increase in the number of cells expressing RANK correlated with the progression of synovial inflammation and clinical disease severity in evolving collagen-induced arthritis (CIA). Interestingly, RANK expression demonstrated a gradient pattern with increased numbers of RANK-positive cells within the synovial infiltrate in areas closer to periosteum and cortical bone. Cells expressing RANK included cells in synovial tissue, bone lining cells on the surface of trabecular bone at sites of erosion, and cells in periosteal areas adjacent to synovial inflammation. In areas where RANK-positive cells were abundant, TRAP-positive, multinucleated osteoclast-like cells were also present at sites of focal bone erosion, suggesting differentiation of synovially derived RANK-positive osteoclast precursor cells into osteoclasts. In addition, TRAP- and cathepsin K-double-positive osteoclast-like cells were detected on the synovial side of cortical bone at sites of early and advanced cortical bone erosion. Sites of RANK expression also correlated well with sites of RANKL expression, and there was a close correlation of the temporal expression of the receptor-ligand pair. Cells expressing RANK increased in abundance with the progression of arthritis in evolving CIA, and sites of RANK-expressing cells correlated with sites of TRAP-positive, multinucleated osteoclast-like cells as well as with sites of RANKL expression. These data support the hypothesis that the RANK/RANKL pathway plays an important role in the process of bone erosion in CIA.

Immunohistochemical localization of receptor activator of nuclear factor kappaB (RANK) and its ligand (RANKL) in human deciduous teeth.

Osteoblasts and osteoclasts are involved in bone formation and resorption. RANK and RANKL [receptor activator of nuclear factor kappaB (ligand)], two cytokine-like proteins of the tumor necrosis factor superfamily, are localized on these bone cells and are crucial for the regulation of osteoclastic cell differentiation from hematopoietic precursors and also for the upregulation of mature osteoclasts mediated by cell-to-cell contact and a subsequent cascade of diverse intracellular signaling processes in the osteoclasts. It was the aim of this study to examine the sites of expression of RANKL and RANK in the corresponding cells of human dental hard and periodontal tissues using immunohistochemical light microscopical methods on tissue sections of 15 paraffin-embedded human deciduous teeth undergoing root resorption. We detected granular cytoplasmic RANKL-immunoreactivity in odontoblasts, pulp fibroblasts, periodontal ligament fibroblasts, and in single odontoclasts, the latter finding suggesting an autocrine/paracrine role. RANK-positive cells were identified as multinucleated odontoclasts localized near the dentine surface in resorption lacunae or as mononucleated precursors. These findings indicate that human dental cells express key mediators of hard tissue resorption and, though the RANK/RANKL-system may not be the sole regulator of tooth root resorption, these factors could at least contribute to this complex process under both physiological and pathological conditions.

The osteoprotegerin/receptor activator of nuclear factor kappaB/receptor activator of nuclear factor kappaB ligand system in cartilage.

The receptor activator of nuclear factor kappaB (RANK) is a member of the tumor necrosis factor receptor family. It is activated by the secreted or cell surface-bound RANK ligand (RANKL). Osteoprotegerin (OPG) is a soluble nonsignaling receptor for RANKL and interferes with RANK activation. This receptor-ligand system regulates the differentiation of osteoclasts and dendritic cells. The present study examined human articular cartilage for the expression of these molecules and the role of RANKL in the regulation of chondrocyte function. Normal and osteoarthritic (OA) human articular cartilage was used for explant tissue culture or for isolation of chondrocytes and cell culture. Expression of RANK, RANKL, and OPG was analyzed by immunohistochemistry, Western blotting, or reverse transcription-polymerase chain reaction. Recombinant RANKL was added to cartilage or chondrocyte cultures, and gene expression, collagenase and nitric oxide production, and NF-kappaB activation were determined. RANK, RANKL, and OPG messenger RNA (mRNA) were expressed in normal cartilage. By immunohistochemistry, RANK, RANKL, and OPG were detected in the superficial zone of normal cartilage. OA cartilage contained increased levels of OPG mRNA, and expression of the 3 proteins extended into the midzone of OA cartilage. OPG was detected by Western blotting, and was increased in response to interleukin-1beta stimulation. OPG, RANK, and RANKL protein were also detected in cultured chondrocytes. Addition of exogenous RANKL did not activate NF-kappaB, induce expression of genes encoding proinflammatory mediators in chondrocytes, or stimulate the production of collagenase and nitric oxide. These results demonstrate the expression of OPG, RANK, and RANKL in cartilage. However, RANKL does not activate human articular chondrocytes.