osteoprotegerin and Osteolysis

Charcot Foot (CF), part of a broader condition known as Charcot Neuro-Osteoarthropathy (CNO), is characterized by neuropathic arthropathy with a progressive alteration of the foot. CNO is one of the most devastating complications in patients with diabetes mellitus and peripheral neuropathy but can also be caused by neurological or infectious diseases. The pathogenesis is multifactorial; many studies have demonstrated the central role of inflammation and the Receptor Activator of NF-κB ligand (RANKL)-Receptor Activator of NF-κB (RANK)-Osteoprotegerin (OPG) pathway in the acute phase of the disease, resulting in the serum overexpression of RANKL. This overexpression and activation of this signal lead to increased osteoclast activity and osteolysis, which is a prelude to bone destruction. The aim of this narrative review is to analyze this signaling pathway in bone remodeling, and in CF in particular, to highlight its clinical aspects and possible therapeutic implications of targeting drugs at different levels of the pathway. Drugs that act at different levels in this pathway are anti-RANKL monoclonal antibodies (Denosumab), bisphosphonates (BP), and calcitonin. The literature review showed encouraging data on treatment with Denosumab, although in a few studies and in small sample sizes. In contrast, BPs have been re-evaluated in recent years in relation to the high possibility of side effects, while calcitonin has shown little efficacy on CNO.

Topics: Bone Density Conservation Agents; Calcitonin; Denosumab; Diabetes Mellitus; Diabetic Foot; Humans; NF-kappa B; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B

More than 2 decades ago, the discovery of osteoprotegerin (OPG) as inhibitor of the receptor of activator of nuclear factor Kb (RANK) ligand (RANKL) revolutionized our understanding of bone biology and oncology. Besides acting as decoy receptor for RANKL, OPG acts as decoy receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). OPG, RANKL, and TRAIL are ubiquitously expressed, stimulating per se pivotal signaling cascades implicated in cancer. In the context of cancer cell-bone cell interactions, cancer cells skew the OPG/RANKL/RANK (RANKL cognate receptor) balance towards bone destruction and tumor growth through favoring the RANKL/RANK interface, circumventing OPG. Numerous preclinical and clinical studies demonstrate the dual role of OPG in cancer: antitumor and tumor-promoting. OPG potentially conveys an antitumor signal through inhibiting the tumor-promoting RANKL signaling-both the osteoclast-dependent and the osteoclast-independent-and the tumor-promoting TRAIL signaling. On the other hand, the presumed tumor-promoting functions of OPG are: (i) abrogation of TRAIL-induced apoptosis of cancer cells; (ii) abrogation of RANKL-induced antitumor immunity; and (iii) stimulation of oncogenic and prometastatic signaling cascades downstream of the interaction of OPG with diverse proteins. The present review dissects the role of OPG in bone oncology. It presents the available preclinical and clinical data sustaining the dual role of OPG in cancer and focuses on the imbalanced RANKL/RANK/OPG interplay in the landmark "vicious cycle" of skeletal metastatic disease, osteosarcoma, and multiple myeloma. Finally, current challenges and future perspectives in exploiting OPG signaling in bone oncology therapeutics are discussed.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone and Bones; Bone Neoplasms; Clinical Trials as Topic; Disease Models, Animal; Drug Screening Assays, Antitumor; Humans; Immunoconjugates; Neoplasm Metastasis; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand

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.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Bone Morphogenetic Proteins; Bone Resorption; Genetic Markers; Humans; Intercellular Signaling Peptides and Proteins; Multiple Myeloma; Osteoclasts; Osteocytes; Osteogenesis; Osteolysis; Osteoprotegerin; RANK Ligand; Wnt Signaling Pathway

Paget's disease was first described more than 150 years ago, but the exact cause is still unknown--genes and the environment are both important. This article explores the basic science and clinical aspects of this intriguing condition.

Topics: Adaptor Proteins, Signal Transducing; Alkaline Phosphatase; Bone and Bones; Bone Density Conservation Agents; Diphosphonates; Humans; Orthopedic Procedures; Osteitis Deformans; Osteolysis; Osteoprotegerin; Osteosclerosis; Radiography; Receptor Activator of Nuclear Factor-kappa B; Sequestosome-1 Protein

Osteolysis and aseptic loosening currently contribute 75 % of implant failures. Furthermore, with over four million joint replacements projected to be performed in the United States annually, osteolysis and aseptic loosening may continue to pose a significant morbidity. This paper reviews the osteolysis cascade leading to osteoclast activation and bone resorption at the biochemical level. Additionally, the metal ion release mechanism from metallic implants is elucidated. Even though metal ions are not the predominating initiator of osteolysis, they do increase the concentration of key inflammatory cytokines that stimulate osteoclasts and prove to be a contributor to osteolysis and aseptic loosening. Osteolysis is a competitive mechanism among a number of biological reactions, which includes debris release, macrophage and osteoclast activation, an inflammatory response as well as metal ion release. Pharmacological therapy for component loosening has also been reviewed. A non-surgical treatment of osteolysis has not been found in the literature and thus may become an area of future research. Even though this research is warranted, comprehensively understanding the immune response to orthopedic implants and their metallic ions, and thus, creating improved prostheses appears to be the most cost-effective approach to decrease the morbidity related to osteolysis and to design implants with greater longevity. The ionic forms, cytokines, toxicity, gene expression, biological effects, and hypersensitivity responses of metallic elements from metal implants are summarized as well.

Topics: Cytokines; Humans; Inflammasomes; Inflammation; Ions; Joint Prosthesis; Macrophages; Metals; Osteoclasts; Osteolysis; Osteoprotegerin; Prosthesis Failure

Squamous cell carcinomas (SCCs) of the gingiva frequently invade the mandible or maxilla; this invasion is associated with a worse prognosis. The bone destruction associated with carcinomal invasion is mediated by osteoclasts rather than directly by the carcinoma. Therefore, if the cellular and molecular mechanisms by which oral SCC regulates bone invasion were known, it could inform the development of new therapeutic targets. Recently, dysregulation of the functional equilibrium in the receptor activator of NF-κB ligand (RANKL)/RANK/osteoprotegerin (OPG) triad has been shown to be responsible for osteolysis associated with the development of malignant tumors in bone sites. Furthermore, the administration of OPG or soluble RANK prevents bone metastasis by cancer cells. In this review, we discuss recent findings indicating that bone invasion by oral SCC is mediated via RANKL/RANK and may be successfully prevented by RANKL inhibition.

Topics: Animals; Bone and Bones; Bone Neoplasms; Carcinoma, Squamous Cell; Humans; Mandible; Mice; Molecular Targeted Therapy; Mouth Neoplasms; NF-kappa B; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B

Bone is one of the most frequent sites of metastasis in patients with malignancies. Up to 90 % of patients with multiple myeloma, and 60 % to 75 % patients with prostate cancer and breast cancer develop bone metastasis at the later stages of their diseases. Bone metastases are responsible for tremendous morbidity in patients with cancer, including severe bone pain, pathologic fractures, spinal cord and nerve compression syndromes, life-threatening hypercalcemia, and increased mortality. Multiple factors produced by tumor cells or produced by the bone marrow microenvironment in response to tumor cells play important roles in activation of osteoclastic bone resorption and modulation of osteoblastic activity in patients with bone metastasis. In this chapter, we will review the genes that play important roles in bone destruction, tumor growth, and osteoblast activity in bone metastasis and discuss the potential therapies targeting the products of these genes to block both bone destruction and tumor growth.

Topics: Activins; Animals; Annexin A2; Bone Neoplasms; Chemokine CCL3; Humans; Interleukin-6; Osteoblasts; Osteolysis; Osteoprotegerin; Parathyroid Hormone-Related Protein; RANK Ligand

Healthy jawbones ensure better tooth anchorage and the ability to masticate and maintain metabolism. This is achieved by a delicate balance between bone formation and resorption in response to functional demands. An imbalance in the expression of receptor activator of nuclear factor kappa-B (RANK) ligand (RANKL) and osteoprotegerin (OPG) or osteoclastogenesis inhibitory factor (OCIF) is believed to be the underlying mechanism of osteolysis in metastases, multiple myelomas, and cancer therapy-induced bone loss in patients. Considered mainly as bone-specific agents to treat postmenopausal osteoporosis, bisphosphonates, in combination with certain chemotherapeutic agents have proved to be effective in prevention of tumor formation and metastatic osteolysis in bone tissue. Osteonecrosis of the jaws associated with them has, however, been of grave concern to the prosthodontist, as it predisposes patients to a bone-deficient basal seat for dental prostheses. This manuscript reviews available information over the past 13 years on possible mechanisms of bone loss, bisphosphonate-induced osteonecrosis of jaw bones, and prosthodontic concerns.

Topics: Alveolar Bone Loss; Bisphosphonate-Associated Osteonecrosis of the Jaw; Bone Density Conservation Agents; Bone Neoplasms; Contraindications; Dental Implants; Dental Prosthesis Retention; Diphosphonates; Humans; Multiple Myeloma; Osteoclasts; Osteolysis; Osteoprotegerin; Parathyroid Hormone-Related Protein; RANK Ligand

It is now widely recognized that the RANKL-RANK-OPG signaling system plays an important role in the differentiation, activation and cell death of osteoclasts. Recently, several genetic disorders involving the RANKL-RANK-OPG system have been discovered. In this article, we would like to show a brief review on a genetic disorder familial expansile osteolysis (FEO) , in which RANK gene mutation was identified, and other genetic disorders of the RANKL-RANK-OPG system.

Topics: Cell Death; Cell Differentiation; Humans; Mutation; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction

Total hip arthroplasty is one of the most commonly performed and successful elective orthopaedic procedures. However, numerous failure mechanisms limit the long-term success including aseptic osteolysis, aseptic loosening, infection, and implant instability. Aseptic osteolysis and subsequent implant failure occur because of a chronic inflammatory response to implant-derived wear particles. To reduce particulate debris and their consequences, implants have had numerous design modifications including high-molecular-weight polyethylene sockets and noncemented implants that rely on bone ingrowth for fixation. Surgical techniques have improved cementation with the use of medullary plugs, cement guns, lavage of the canal, pressurization, centralization of the stem, and reduction in cement porosity. Despite these advances, aseptic osteolysis continues to limit implant longevity. Numerous proinflammatory cytokines, such as interleukin-1, interleukin-6, tumor necrosis factor-alpha, and prostaglandin E2, have proosteoclastogenic effects in response to implant-derived wear particles. However, none of these cytokines represents a final common pathway for the process of particle-induced osteoclast differentiation and maturation. Recent work has identified the fundamental role of the RANKL-RANK-NF-kappaB pathway not only in osteoclastogenesis but also in the development and function the immune system. Thus, the immune system and skeletal homeostasis may be linked in the process of osteoclastogenesis and osteolysis.

Topics: Arthroplasty, Replacement, Hip; Cytokines; Hip Prosthesis; Humans; Osteolysis; Osteoprotegerin; Prosthesis Design; Prosthesis Failure; Receptor Activator of Nuclear Factor-kappa B

Bone metastasis is often the penultimate harbinger of death for many cancer patients. Bone metastases are often associated with fractures and severe pain resulting in decreased quality of life. Accordingly, effective therapies to inhibit the development or progression of bone metastases will have important clinical benefits. To achieve this goal understanding the mechanisms through which bone metastases develop and progress may provide targets to inhibit the metastases. In the past few years, there have been advances in both understanding the mechanisms through which bone metastases develop and how they impact bone remodeling. Additionally, gains in promising clinical strategies to target bone metastases have been developed. In this prospectus, we will discuss some of these advances.

Topics: Antineoplastic Agents; Bone Neoplasms; Breast Neoplasms; Diphosphonates; Female; Humans; Male; Neoplasm Metastasis; Osteolysis; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand

Identification of the RANKL/OPG/RANK/NF-kB (receptor activator of nuclear factor kappa-B ligand / osteoprotegerin) signaling pathway as the major regulatory system for osteoclastogenesis began with discovery of these ligands and receptors in the tumor necrosis factor (TNF) superfamily. Subsequently, genetically altered mice revealed physiologic roles for these proteins in bone biology. However, full appreciation of their significance for the human skeleton came from clinical characterization of several extremely rare, heritable disorders followed by discovery of their genetic bases. Familial expansile osteolysis (FEO) is an autosomal dominant disorder featuring constitutive activation of RANK due to an 18-bp tandem duplication in its gene (TNFRSF11A). A similar, 27-bp duplication causes what has been called a familial form of early-onset Paget's disease of bone (PDB2). Expansile skeletal hyperphosphatasia (ESH) is allelic to FEO and PDB2 and involves a 15-bp tandem duplication in TNFRSF11A. Autosomal recessive inheritance of deactivating mutations of the gene encoding OPG (TNFRSF11B) causes most cases of juvenile Paget disease. These disorders feature high bone turnover, deafness during early childhood, "idiopathic external lysis" of adult teeth, and sometimes focal lesions in appendicular bones that mimic active PDB. Biochemical markers indicate rapid skeletal remodeling. In FEO, osteolysis progresses to fat-filled bone rather than to osteosclerosis. Antiresorptive therapy with bisphosphonates can be effective for each disorder.

Topics: Carrier Proteins; Genetic Variation; Glycoproteins; Humans; Membrane Glycoproteins; NF-kappa B; Osteitis Deformans; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Signal Transduction

Paget's disease of bone (PDB) is a common disorder in which focal abnormalities of increased bone turnover lead to complications such as bone pain, deformity, pathological fractures, and deafness. PDB has a strong genetic component and several susceptibility loci for the disease have been identified by genome-wide scans. Mutations that predispose individuals to PDB and related disorders have been identified in four genes. The rare PDB-like syndromes of familial expansile osteolysis, early-onset familial PDB, and expansile skeletal hyperphosphatasia are caused by insertion mutations in TNFRSF11A, which encodes receptor activator of nuclear factor (NF)kappaB (RANK)-a critical regulator of osteoclast function. Inactivating mutations in TNFRSF11B, which encodes osteoprotegerin (a decoy receptor for RANK ligand) cause idiopathic hyperphosphatasia, and polymorphisms in this gene seem to increase the risk for classical PDB. Mutations of the sequestosome 1 gene (SQSTM1), which encodes an important scaffold protein in the NFkappaB pathway, are a common cause of classical PDB. The rare syndrome of hereditary inclusion body myopathy, PDB, and fronto-temporal dementia is caused by mutations in the valosin-containing protein (VCP) gene. This gene encodes VCP, which has a role in targeting the inhibitor of NFkappaB for degradation by the proteasome. Several additional genes for PDB remain to be discovered, and it seems likely that they will also involve the RANK-NFkappaB signaling pathway or components of the proteasomal processing of this pathway, underscoring the critical importance of this signaling pathway in bone metabolism and bone disease.

Topics: Adaptor Proteins, Signal Transducing; Adenosine Triphosphatases; Carrier Proteins; Cell Cycle Proteins; Genetic Predisposition to Disease; Glycoproteins; Humans; Loss of Heterozygosity; Membrane Glycoproteins; Myositis, Inclusion Body; NF-kappa B; Osteitis Deformans; Osteolysis; Osteoprotegerin; Polymorphism, Genetic; Proteins; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Sequestosome-1 Protein; Valosin Containing Protein

The recent identification of key molecular protagonists involved in osteoclast biology has led to the development of new therapeutic approaches of osteolytic diseases using biological molecules which could compete with bisphosphonate therapy.

Topics: Animals; Diphosphonates; Drug Design; Glycoproteins; Humans; Integrin alphaVbeta3; Molecular Mimicry; Osteolysis; Osteoprotegerin; Peptides; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Signal Transduction

One of the most characteristic features of multiple myeloma is the development of osteolytic bone lesions. Myeloma-associated bone disease is caused by an increase in osteoclastic bone resorption and a decrease in osteoblastic new bone formation. Insight into the molecular mechanisms of osteoclastogenesis has been provided by the detection of receptor activator of NF-kappaB ligand (RANKL), its specific receptor (RANK) and its decoy receptor antagonist osteoprotegerin (OPG). The RANK signaling system is abnormally regulated in multiple myeloma and targeting this system may ameliorate myeloma bone disease. Less is known about the development of osteoblastic dysfunction, and further knowledge about the interaction between myeloma cells and osteoblasts is required. The aim of this review is to focus on the principles of bone biology for a better understanding of the development of myeloma bone disease and to identify possible therapeutic targets.

Topics: Bone Remodeling; Bone Resorption; Carrier Proteins; Cell Differentiation; Diphosphonates; Glycoproteins; Humans; Membrane Glycoproteins; Multiple Myeloma; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Osteoprotegerin (OPG), a member of the TNF-receptor family expressed by osteoblasts, has documented effects on the regulation of bone metabolism. OPG inhibits bone resorption and binds with strong affinity to its ligand RANKL, thereby preventing RANKL from binding to its receptor RANK. This system is regulated by calcium-modifying hormones. OPG may also be pivotal in modulating the immune system. RANKL-deficient mice exhibit both severe immunological abnormalities and osteopetrosis, and activated T cells express RANKL mRNA. RANKL secretion by activated T cells may induce osteoclastogenesis via a mechanism enhanced by several cytokines (TNF-alpha, IL-1, and IL-17) that promote both inflammation and bone resorption. Conversely, this mechanism is inhibited by OPG, IL-4, and IL-10, which have antiinflammatory effects and inhibit osteoclast formation. Activated T cells in the rheumatoid synovium express RANKL. Synoviocytes can differentiate to osteoclast-like cells under specific conditions, particularly when they are cultured with M-CSF and RANKL. Thus, the bony erosions seen in RA may result from RANKL/RANK system activation by activated T cells. This raises the possibility that OPG therapy to block this mechanism might prove beneficial in patients with RA.

Topics: Animals; Arthritis, Rheumatoid; Glycoproteins; Humans; Inflammation; Mice; Osteoblasts; Osteolysis; Osteoprotegerin; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Implants, particularly joint replacement prostheses, are one of the great success stories of modern medicine. However, too many implants fail prematurely, mainly due to aseptic bone loss around the implant. This paper reviews our current understanding of the role of osteoclasts in this peri-implant bone lysis. Prosthetic particles, often produced by articulating prostheses, are one of the major causes of elevated osteoclast lysis of peri-implant bone. Over the past decade there have been major advances in our understanding of the factors that regulate osteoclast activity, many of which were found to be important in osteoclast formation and activity in the peri-implant tissues. These factors are targets of a number of recently developed drugs that have been used successfully to prevent and treat peri-implant bone lysis in experimental models. Treatments such as these are being used in a number of bone loss pathologies in humans and have the potential for successful treatment of peri-implant osteolysis. In addition, understanding how different biomaterials influence the expression of key osteoclastogenic factors may allow us to select biomaterials for implantation that will last the lifetime of the recipient.

Topics: Animals; Arthroplasty, Replacement, Hip; Biocompatible Materials; Bone and Bones; Carrier Proteins; Glycoproteins; Humans; Membrane Glycoproteins; Models, Biological; Osseointegration; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Diabetic neuropathy is associated with osteopenia and calcification of vascular smooth muscle cells. These changes are most marked in patients with acute neuropathic osteoarthropathy (Charcot foot), in which osteopenia is universal and the prevalence of vascular calcification exceeds 90%. While it has been thought that both osteopenia and vascular calcification may be linked to sympathetic denervation with increased peripheral limb perfusion, the cellular mechanism was not clear. However, the recent recognition that the receptor activator of nuclear factor kappa B ligand (RANK-L)/osteoprotegerin (OPG) signalling pathway is central to the processes regulating bone turnover in a wide variety of medical conditions has raised the possibility that the same cytokines may be involved in the osteolysis which accompanies diabetic neuropathy. This is made more likely by the realisation that the RANK-L/OPG pathway is also thought to mediate the calcification of vascular smooth muscle cells in coronary and peripheral vascular disease. The circumstantial evidence underpinning this hypothesis is reviewed here, and it is suggested that the unregulated activation of RANK-L-mediated effects on bone and arteries may be triggered by the loss of nerve-derived peptides, e.g. calcitonin gene-related peptide, which normally exert a moderating influence on the pathway.

Topics: Calcitonin Gene-Related Peptide; Carrier Proteins; Diabetic Neuropathies; Gait Disorders, Neurologic; Glycoproteins; Humans; Membrane Glycoproteins; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Signal Transduction

Elucidation of the receptor activator of nuclear factor kappa B (RANK), its ligand (RANKL) and osteoprotegerin (OPG) as the final effectors of bone resorption has transformed our understanding of metabolic bone diseases and revealed novel therapeutic targets. Activation of the RANK-RANKL signaling pathway is directly responsible for dramatic focal erosions that are observed in inflammatory arthritis and aseptic loosening of orthopaedic implants. While these conditions share many features common to all metabolic bone disorders (e.g., osteoclastic resorption), they exhibit several unique properties, which are highlighted in this review. Most important is the relative inability of bisphosphonate therapy to inhibit osteolysis in joint inflammation and periprosthetic joint loosening and the unexpected effectiveness of anti-cytokine therapy in both rheumatoid and psoriatic arthritis. Herein, we provide a review of the role of RANK, RANKL and OPG in erosive arthritis and periprosthetic osteolysis and discuss the potential of anti-RANKL therapy for these conditions.

Topics: Animals; Arthritis; Carrier Proteins; Glycoproteins; Humans; Membrane Glycoproteins; Osteolysis; Osteoprotegerin; Prosthesis Failure; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Over the past decade there have been major advances in our understanding of the factors that regulate osteoclast formation and activity. It is now apparent that receptor activator NFkappaB (RANK), its ligand, RANKL (also known as TRANCE, osteoclast differentiation factor and osteoprotegerin (OPG) ligand) and the natural RANKL inhibitor, OPG, are the key factors regulating osteoclast formation in normal bone physiology. The molecular interactions of these molecules regulate osteoclast formation and subsequent bone loss in disease and there is now strong evidence that the bone loss associated with inflammatory diseases, such as rheumatoid arthritis, periodontal disease and peri-implant loosening, is regulated by the action of RANK, RANKL, and OPG. These molecules are targets for the pharmacological regulation of severe bone loss in several common inflammatory diseases.

Topics: Animals; Arthritis, Rheumatoid; Bone Resorption; Carrier Proteins; Cell Differentiation; Glycoproteins; Humans; Ligands; Membrane Glycoproteins; NF-kappa B; Osteitis; Osteoclasts; Osteolysis; Osteoprotegerin; Periodontitis; Prosthesis Failure; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Multiple myeloma (MM) is a plasma cell malignancy characterized by the high capacity to induce osteolytic bone lesions that mainly result from an increased bone resorption related to the stimulation of osteoclast recruitment and activity. Although it is known that myeloma cells induce osteoclastic bone resorption, the biological mechanisms involved in the pathophysiology of MM-induced bone resorption have been unclear for several years. Recently, new data seem to elucidate which mechanism is critically involved in the activation of osteoclastic cells in MM. The critical osteoclastogenetic factor RANKL and its soluble antagonist osteoprotegerin (OPG) are the major candidates in the pathophysiology of MM bone disease. Human MM cells induce an imbalance in the RANKL/OPG ratio in the bone marrow environment that triggers the osteoclast formation and activation leading to bone destruction. The role or RANKL/OPG system and other osteoclast stimulating factors in the pathophysiology of MM bone disease are summarized in this update.

Topics: Animals; Bone Marrow; Bone Resorption; Carrier Proteins; Cell Differentiation; Cell Line, Tumor; Chemokine CCL4; Culture Media, Conditioned; Cytokines; Glycoproteins; Humans; Interleukin-6; Interleukin-7; Macrophage Inflammatory Proteins; Membrane Glycoproteins; Mice; Multiple Myeloma; Neoplasm Proteins; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Stromal Cells; T-Lymphocyte Subsets

Osteoclasts are the only cells capable of resorbing mineralised bone, dentine and cartilage. Osteoclasts act in close concert with bone forming osteoblasts to model the skeleton during embryogenesis and to remodel it during later life. A number of inherited human conditions are known that are primarily caused by a defect in osteoclasts. Most of these are rare monogenic disorders, but others, such as the more common Paget's disease, are complex diseases, where genetic and environmental factors combine to result in the abnormal osteoclast phenotype. Where the genetic defect gives rise to ineffective osteoclasts, such as in osteopetrosis and pycnodysostosis, the result is the presence of too much bone. However, the phenotype in many osteoclast diseases is a combination of osteosclerosis with osteolytic lesions. In such conditions, the primary defect is hyperactivity of osteoclasts, compensated by a secondary increase in osteoblast activity. Rapid progress has been made in recent years in the identification of the causative genes and in the understanding of the biological role of the proteins encoded. This review discusses the known osteoclast diseases with particular emphasis on the genetic causes and the resulting osteoclast phenotype. These human diseases highlight the critical importance of specific proteins or signalling pathways in osteoclasts.

Topics: Animals; Bone and Bones; Bone Diseases; Bone Resorption; Cell Differentiation; Glycoproteins; Humans; Osteitis Deformans; Osteoclasts; Osteolysis; Osteopetrosis; Osteoprotegerin; Osteosclerosis; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow (BM) and characterized by a high capacity for bone destruction. Almost all patients with MM have early osteolytic lesions, which result mainly from increased bone resorption related to stimulation of osteoclast recruitment and activity in the immediate vicinity of myeloma cells. The recent discovery of Osteoprotegerin (OPG) and the subsequent identification of its ligand RANKL have provided new insights in the regulation of osteoclastogenesis. The ratio OPG/RANKL is critical for the regulation of bone remodeling maintaining the balance between osteoblastic and osteoclastic activity. This review summarizes the new concept that myeloma cells induce in bone environment an imbalance in the OPG/RANKL system responsible for osteolysis observed in patients. Indeed, myeloma cells increase in bone environment the expression of the potent osteoclastogenic factor RANKL and decrease the osteoprotective factor OPG production. Biological mechanisms involved in these processes are discussed. Furthermore, the chemokines MIP-1alpha and MIP-1beta belonging to the RANTES family are potent osteoclastogenic factors produced by myeloma cells and participate in myeloma-associated bone disease. These data open new avenues for the treatment of bone disease in MM and highlight the promising therapeutical interest of RANKL inhibitors (OPG and RANK-Fc) and MIP-1 inhibitors in the management of myeloma-associated osteolysis, besides bisphosphonates.

Topics: Animals; Bone Remodeling; Carrier Proteins; Chemokine CCL3; Chemokine CCL4; Diphosphonates; Drug Design; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Hypercalcemia; Macrophage Inflammatory Proteins; Membrane Glycoproteins; Mice; Multiple Myeloma; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Fusion Proteins

Extensive osteolysis adjacent to implants is often associated with wear particles of prosthetic material. We have investigated if RANKL, also known as osteoprotegerin ligand, osteoclast differentiation factor or TRANCE, and its natural inhibitor, osteoprotegerin (OPG), may be important in controlling this bone loss. Cells isolated from periprosthetic tissues containing wear particles expressed mRNA encoding for the pro-osteoclastogenic molecules, RANKL, its receptor RANK, monocyte colony-stimulating factor (M-CSF), interleukin (IL)-1beta, tumour necrosis factor (TNF)alpha, IL-6, and soluble IL-6 receptor, as well as OPG. Osteoclasts formed from cells isolated from periprosthetic tissues in the presence and absence of human osteoblastic cells. When osteoclasts formed in the absence of osteoblastic cells, markedly higher levels of RANKL mRNA relative to OPG mRNA were expressed. Particles of prosthetic materials also stimulated human monocytes to express osteoclastogenic molecules in vitro. Our results suggest that ingestion of prosthetic wear particles by macrophages results in expression of osteoclast-differentiating molecules and the stimulation of macrophage differentiation into osteoclasts.

Topics: Carrier Proteins; Cells, Cultured; Cytokines; Glycoproteins; Interleukin-6; Macrophage Colony-Stimulating Factor; Macrophages; Membrane Glycoproteins; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Necrosis Factor-alpha

There is currently no cure for aseptic loosening (AL) of total joint replacement (TJR) except surgical revision. The purpose of this study was to determine whether oral EM could improve the periprosthetic tissue profiles and reduce serum cytokine production in AL patients who are candidates for surgical revision. We recruited 32 AL patients. AL patients were treated with either EM (600 mg/day, n=18) or placebo (n=14) daily, started one month before surgery and ending on the day of surgery. Blood samples were obtained before EM treatment and during surgery. Periprosthetic tissues and joint fluids were collected during surgery. Our results demonstrate that oral EM reduces the inflammation of periprosthetic tissues, as manifested by the reduction of the numbers of infiltrating cells, CD68+ macrophages, RANKL+ cells, and TRAP+ cells. Remarkable decreases of TNFalpha (9.6-fold), IL-1beta (21.2-fold), and RANKL (76-fold) gene transcripts were observed in periprosthetic tissues of patients treated with oral EM. Serum levels of both TNFalpha and (to a lesser extent) IL-1beta were significantly reduced following EM treatment (p<0.05). Our results suggest that EM represents a biological cure or prevention for those patients who might need repeated revision surgeries and/or show the early signs of progressive osteolysis after TJR.

Topics: Administration, Oral; Aged; Anti-Bacterial Agents; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Cytokines; Double-Blind Method; Enzyme-Linked Immunosorbent Assay; Erythromycin; Female; Gene Expression; Gene Expression Profiling; Humans; Immunohistochemistry; Inflammation; Joint Prosthesis; Leukocytes, Mononuclear; Male; Middle Aged; Osteolysis; Osteoprotegerin; Prosthesis Failure; RANK Ligand; Reoperation; Reverse Transcriptase Polymerase Chain Reaction

Aseptic bone loss adjacent to orthopedic joint implants is a common cause of joint implant failure in humans. This study investigates the expression of key regulators of osteoclast formation, receptor activator NFkappaB (RANK), Receptor activator of NFkappaB ligand (RANKL) and osteoprotegerin (OPG), in the peri-implant tissues of patients with osteolysis compared with levels in synovial tissues from osteoarthritic and healthy subjects. Immunohistochemical studies demonstrated that significantly higher levels of RANKL protein (p<0.05) were found in the peri-implant tissues of patients with implant failure than in similar tissues from osteoarthritic and healthy subjects. In contrast, OPG protein levels were similar in all tissues. RANKL, expressed as mRNA and protein, was predominantly associated with cells containing wear particles. Dual labeling studies showed that the cells expressing RANKL protein were macrophages. In situ hybridization studies confirmed that mRNA encoding for these proteins is also expressed by cells in the peri-implant tissues. In addition, RANK mRNA was expressed in cells that contained wear particles. These findings show that abnormally high levels of RANKL are expressed in peri-implant tissues of patients with prosthetic loosening and that these abnormal levels of RANKL may significantly contribute to aseptic implant loosening.

Topics: Adult; Aged; Aged, 80 and over; Carrier Proteins; Female; Foreign-Body Reaction; Glycoproteins; Humans; Male; Membrane Glycoproteins; Middle Aged; NF-kappa B; Osteoarthritis; Osteoclasts; Osteolysis; Osteonecrosis; Osteoprotegerin; Prosthesis Failure; Prosthesis-Related Infections; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Aseptic loosening caused by periprosthetic osteolysis (PPO) is the main reason for the primary artificial joint replacement. Inhibition of inflammatory osteolysis has become the main target of drug therapy for prosthesis loosening. MiR-106b is a newly discovered miRNA that plays an important role in tumour biology, inflammation and the regulation of bone mass. In this study, we analysed the in vivo effect of miR-106b on wear debris-induced PPO. A rat implant loosening model was established. The rats were then administrated a lentivirus-mediated miR-106b inhibitor, miR-106b mimics or an equivalent volume of PBS by tail vein injection. The expression levels of miR-106b were analysed by real-time PCR. Morphological changes in the distal femurs were assessed via micro-CT and histopathological analysis, and cytokine expression levels were examined via immunohistochemical staining and ELISA. The results showed that treatment with the miR-106b inhibitor markedly suppressed the expression of miR-106b in distal femur and alleviated titanium particle-induced osteolysis and bone loss. Moreover, the miR-106b inhibitor decreased TRAP-positive cell numbers and suppressed osteoclast formation, in addition to promoting the activity of osteoblasts and increasing bone formation. MiR-106b inhibition also significantly regulated macrophage polarization and decreased the inflammatory response as compared to the control group. Furthermore, miR-106b inhibition blocked the activation of the PTEN/PI3K/AKT and NF-κB signalling pathways. Our findings indicated that miR-106b inhibition suppresses wear particles-induced osteolysis and bone destruction and thus may serve as a potential therapy for PPO and aseptic loosening.

Topics: Animals; Bone and Bones; Bone Resorption; Cell Count; Cell Polarity; Cytokines; Inflammation; Kidney; Liver; Macrophages; Male; MicroRNAs; NF-kappa B; Osteoclasts; Osteogenesis; Osteolysis; Osteoprotegerin; Phosphatidylinositol 3-Kinases; Prostheses and Implants; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; RANK Ligand; Rats, Sprague-Dawley; Signal Transduction; Titanium

Many species of theBauhinia genus have been widely used in folk medicine as analgesic, anti-inflammatory and antioxidant agents. (-)-Fisetinidol palmitate is a semi-syntetic flavonoid obtained from the ethanolic extract of the stem of Bauhinia pulchella. This study aimed to evaluate the antiresorptive effect of the semi-syntetic (-)-fisetinidol palmitate in ligature-induced periodontitis in rats. Also, it evaluated the mechanism of action of (-)-fisetinidol palmitate and its toxicity.. Periodontitis was inducedvia a nylon thread ligature (3.0) around the second upper left molars. Rats were treated (oral gavage) once a day for 11 days with (-)-fisetinidol palmitate (0.01 or 0.1 mg/kg) or saline vehicle.. (-)-Fisetinidol palmitate (0.1 mg/kg) reduced alveolar bone loss, increased bone alkaline phosphatase (BALP), superoxide dismutase (SOD), and catalase (CAT) activity; also, it decreased IL1-β, IL-8/CINC-1, nitrite/nitrate levels and myeloperoxidase activity. (-)-Fisetinidol palmitate reduced the mRNA levels of IL1-β, IL-6, RANK, and RANK-L, while it increased the OPG ones. No statistical differences (P > 0.05) were observed in the transaminases (ALT, AST) and Total Alkaline Phosphatase (TALP) levels among groups.. Fisetinidol palmitate did not result in any signs of toxicity and had anti-resorptive effects in a pre-clinical trial of periodontitis, showing antioxidant activity with the involvement of the RANK/RANKL/OPG pathway.

Topics: Alveolar Bone Loss; Animals; Antioxidants; Bauhinia; Cytokines; Flavonoids; Osteolysis; Osteoprotegerin; Oxidative Stress; Periodontitis; Phytochemicals; Plant Stems; RANK Ligand; Rats; Rats, Wistar

Aseptic loosening caused by wear particles is one of the common complications after total hip arthroplasty. We investigated the effect of the recombinant protein ephB4-Fc (erythropoietin-producing human hepatocellular receptor 4) on wear particle-mediated inflammatory response. In vitro, ephrinB2 expression was analyzed using siRNA-NFATc1 (nuclear factor of activated T-cells 1) and siRNA-c-Fos. Additionally, we used Tartrate-resistant acid phosphatase (TRAP) staining, bone pit resorption, Enzyme-linked immunosorbent assay (ELISA), as well as ephrinB2 overexpression and knockdown experiments to verify the effect of ephB4-Fc on osteoclast differentiation and function. In vivo, a mouse skull model was constructed to test whether the ephB4-Fc inhibits osteolysis and inhibits inflammation by micro-CT, H&E staining, immunohistochemistry, and immunofluorescence. The gene expression of ephrinB2 was regulated by c-Fos/NFATc1. Titanium wear particles activated this signaling pathway to the promoted expression of the ephrinB2 gene. However, ephrinB2 protein can be activated by osteoblast membrane receptor ephB4 to inhibit osteoclast differentiation. In in vivo experiments, we found that ephB4 could regulate Ti particle-mediated imbalance of OPG/RANKL, and the most important finding was that ephB4 relieved the release of proinflammatory factors. The ephB4-Fc inhibits wear particle-mediated osteolysis and inflammatory response through the ephrinB2/EphB4 bidirectional signaling pathway, and ephrinB2 ligand is expected to become a new clinical drug therapeutic target.

Topics: Actins; Animals; Bone Resorption; Cell Differentiation; Ephrin-B2; Female; Humans; Inflammation Mediators; Interleukin-1beta; Interleukin-6; Mice, Inbred C57BL; NFATC Transcription Factors; Osteoblasts; Osteoclasts; Osteogenesis; Osteolysis; Osteoprotegerin; Proto-Oncogene Proteins c-fos; RANK Ligand; Receptor, EphB4; Recombinant Proteins; Signal Transduction; Titanium; Tumor Necrosis Factor-alpha

Periprosthetic osteolysis (PIO) caused by wear particles is the main cause of implant failure, which is regulated by nuclear factor κ B receptor activator ligand (RANKL)/osteoprotegerin (OPG) system. At present, there is a lack of effective drugs to prevent or treat PIO. Previous studies have confirmed that DNA methylation is closely related to postmenopausal osteoporosis and can affect the expression of OPG and RANKL. However, the relationship between DNA methylation and PIO is not clear. In this study, we investigated the inhibitory effect of 5-Aza-2-deoxycytidine (AzadC) on osteolysis induced by titanium particles in a mouse model. This inhibition mechanism is achieved by changing the ratio of RANKL/OPG in the osteolysis model. In conclusion, there is a relationship between DNA methylation and PIO. AzadC has a certain inhibitory effect on osteolysis induced by titanium particles. Regulating DNA methylation may be a new way to treat PIO. Our findings lay a foundation for epigenetic understanding and intervention of osteolysis.

Topics: Animals; Base Sequence; Blotting, Western; CpG Islands; Decitabine; DNA Methylation; Gene Expression; Male; Mice, Inbred BALB C; Osteolysis; Osteoprotegerin; Particle Size; RANK Ligand; Reverse Transcriptase Polymerase Chain Reaction; Skull; Titanium; X-Ray Microtomography

The osteoclast bone resorption is critical in aseptic loosening after joint replacement. The balance between activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) is considered to play a central role in osteoclast maturation. Fibroblasts from the periprosthetic membrane express RANKL and promote osteoclast formation. Studies have demonstrated that naringin inhibited osteoclastogenesis and wear particle-induced osteolysis. In this study, the naringin-induced OPG/RANKL effects and its underlying mechanism were studied in fibroblasts from periprosthetic membrane.. Fibroblasts were isolated from the periprosthetic membrane during hip arthroplasty for revision due to aseptic loosening. Fibroblasts were cultured and treated with or without naringin and DKK-1 (the classical inhibitor of Wnt/β-catenin signaling pathway). OPG and RANKL mRNA and protein levels, gene expression of β-catenin, and cyclin D1, which participate in the Wnt signaling pathway, were examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay.. The mRNA and protein levels of OPG were enhanced by naringin in a dose-dependent manner compared to that of the non-treated control. In contrast, naringin did not affect the expression of RANKL. Importantly, DKK-1 attenuated OPG expression in fibroblasts under naringin treatment. Moreover, naringin stimulated the gene expression of β-catenin and cyclin D1 in fibroblasts, and the effect could be inhibited by DKK-1.. The results indicated that naringin enhanced OPG expression through Wnt/β-catenin signaling pathway in fibroblasts from periprosthetic membrane, which may be useful to inhibit periprosthetic osteolysis during aseptic loosening after total joint arthroplasty.

Topics: Aged; Aged, 80 and over; Arthroplasty, Replacement, Hip; beta Catenin; Bone Resorption; Cells, Cultured; Female; Fibroblasts; Flavanones; Gene Expression; Humans; Interosseous Membrane; Male; Middle Aged; Osteoclasts; Osteogenesis; Osteolysis; Osteoprotegerin; Prosthesis Failure; RANK Ligand; RNA, Messenger; Signal Transduction; Wnt Proteins

Metformin, an anti-hyperglycemic agent used for type 2 diabetes, has recently been found to have more effects apart from glucose regulation. We found that, in ultra-high-molecular-weight polyethylene particle-induced osteolysis mouse models, metformin had bone protect property and reduced the negative regulator of bone formation sclerostin (SOST) and Dickkopf-related protein 1 (DKK1), and increased osteoprotegerin (OPG) secretion and the ratio of OPG/Receptor Activator for Nuclear Factor-κB Ligand (RANKL). In vitro, we established a 3D co-culture system in which metformin affects osteoblasts and osteoclasts through mature osteocytes secretion. Metformin (50 μM) significantly decreased SOST and DKK1 mRNA expression, stimulating alkaline phosphatase activity and proliferation of osteoblast, and increased OPG secretion and the ratio of OPG/RANKL, inhibiting osteoclastogenesis. Moreover, the effect on OPG was reversed by adenosine 5'-monophosphate-activated protein kinase inhibitor, Compound C. Our finding suggests that metformin induces differentiation and mineralization of osteoblasts, while inhibits osteoclastogenesis via mature osteocytes secretion. Therefore, the drug might be beneficial for not only diabetes but also in other bone disorders by acting on mature osteocytes.

Topics: Adaptor Proteins, Signal Transducing; Adenylate Kinase; Animals; Bone and Bones; Cell Differentiation; Cell Proliferation; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Glycoproteins; Inflammation; Intercellular Signaling Peptides and Proteins; Male; Metformin; Mice, Inbred C57BL; Models, Biological; Organ Size; Osteocytes; Osteogenesis; Osteolysis; Osteoprotegerin; Phosphorylation; Polyethylenes; Protective Agents; RANK Ligand; Skull

Periprosthetic asepteic loosening, caused by wear debris, is one of the most severe complications, generally resulting in implant failure. Extensive osteoclast formation and activation are considered as the cause for periprosthetic osteolysis. However, few approaches have been approved to be used for preventing early-stage periprosthetic osteolysis. In this study, we investigated the preventive effects of CEP on titanium particles-induced osteolysis in a murine calvaria model. This inhibitory effect was confirmed to be realized by attenuating osteoclastogenesis in vivo. In addition, CEP markedly reduced wear particles-induced elevation of receptor activator of nuclear factor kappa B ligand (RANKL)/Osteoprotegerin (OPG) ratio in vivo. In conclusion, these data concluded that CEP demonstrated a preventive effect of CEP on titanium particles induced osteolysis, suggesting that CEP might be a novel therapeutic for periprosthesis loosening.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Benzylisoquinolines; Bone-Anchored Prosthesis; Bone-Implant Interface; Cathepsin D; Disease Models, Animal; Gene Expression Regulation; Male; Mice; NFATC Transcription Factors; Osteoclasts; Osteogenesis; Osteolysis; Osteoprotegerin; Prosthesis Failure; RANK Ligand; Skull; Tartrate-Resistant Acid Phosphatase; Titanium

Currently, there are no medications available to treat aseptic loosening of orthopedic implants. Using osteoprotegerin fusion protein (OPG-Fc), we previously blocked instability-induced osteoclast differentiation and peri-prosthetic osteolysis. Wnt/β-catenin signaling, which regulates OPG secretion from osteoblasts, also modulates the bone tissue response to mechanical loading. We hypothesized that activating Wnt/β-catenin signaling by inhibiting glycogen synthase kinase-3β (GSK-3β) would reduce instability-induced bone loss through regulation of both osteoblast and osteoclast differentiation. We examined effects of GSK-3β inhibition on regulation of RANKL and OPG in a rat model of mechanical instability-induced peri-implant osteolysis. The rats were treated daily with a GSK-3β inhibitor, AR28 (20 mg/kg bw), for up to 5 days. Bone tissue and blood serum were assessed by qRT-PCR, immunohistochemistry, and ELISA on days 3 and 5, and by micro-CT on day 5. After 3 days of treatment with AR28, mRNA levels of β-catenin, Runx2, Osterix, Col1α1, and ALP were increased leading to higher osteoblast numbers compared to vehicle-treated animals. BMP-2 and Wnt16 mRNA levels were downregulated by mechanical instability and this was rescued by GSK-3β inhibition. Osteoclast numbers were decreased significantly after 3 days of GSK-3β inhibition, which correlated with enhanced OPG mRNA expression. This was accompanied by decreased serum levels of TRAP5b on days 3 and 5. Treatment with AR28 upregulated osteoblast differentiation, while osteoclastogenesis was blunted, leading to increased bone mass by day 5. These data suggest that GSK-3β inactivation suppresses osteolysis through regulating both osteoblast and osteoclast differentiation in a rat model of instability-induced osteolysis.

Topics: Alkaline Phosphatase; Animals; beta Catenin; Bone Morphogenetic Protein 2; Bone Plates; Cell Differentiation; Cell Proliferation; Collagen Type I; Collagen Type I, alpha 1 Chain; Core Binding Factor Alpha 1 Subunit; Disease Models, Animal; Gene Expression Regulation; Glycogen Synthase Kinase 3 beta; Male; Osteoblasts; Osteoclasts; Osteogenesis; Osteolysis; Osteoprotegerin; Prosthesis Failure; Prosthesis Implantation; Protein Kinase Inhibitors; RANK Ligand; Rats, Sprague-Dawley; RNA, Messenger; Tartrate-Resistant Acid Phosphatase; Tibia; Time Factors; Transcription Factors; Wnt Signaling Pathway

The present study aimed to investigate the effects of strontium ranelate (SR), an anti‑osteoporotic drug, on osteolysis in an experimental mouse model of aseptic loosening. A total of 45 female C57BL/6J mice each received implantation of one titanium alloy pin into the tibia, followed by intraarticular injection of titanium particles. One week following surgery, mice were randomly divided into three groups: Control group (no additional treatment), SR625 group (treated with SR at a dose of 625 mg/kg/day), and SR1800 group (treated with SR at a dose of 1,800 mg/kg/day). SR was administered via oral gavage once every day for 12 weeks. Micro‑computed tomography scanning and hematoxylin/eosin staining were used to assess osteolysis around the prosthesis. Immunohistochemistry and reverse transcription-quantitative polymerase chain reaction analysis were used to measure the expression of receptor activator of nuclear factor‑κB ligand (RANKL) and osteoprotegerin (OPG). Compared with the control, the SR625 and SR1800 groups exhibited a significantly increased pulling force of the titanium alloy pin. Bone volume and the bone surface/volume ratio in the periprosthetic tissue were significantly increased in the SR‑treated groups. Significant differences were observed between the SR1800 group and control group with respect to trabecular thickness and trabecular number. Mechanistically, SR downregulated the expression of RANKL and upregulated the expression of OPG in the periprosthetic tissue. In addition, SR was observed to inhibit wear particle‑associated osteolysis in a dose‑dependent manner. In conclusion, the present data illustrated that SR inhibited titanium particle‑induced osteolysis in vivo.

Topics: Administration, Oral; Alloys; Animals; Biomarkers; Bone Density Conservation Agents; Bone-Implant Interface; Dose-Response Relationship, Drug; Female; Gene Expression; Mice; Mice, Inbred C57BL; Osteolysis; Osteoprotegerin; Particulate Matter; Prostheses and Implants; Prosthesis Failure; RANK Ligand; Thiophenes; Tibia; Titanium

We examined the effects of recombinant human osteoclastogenesis inhibitory factor (hOCIF) on osteolysis, proliferation of mammary tumor cells, and induction of cancer stem cells (CSCs) in the tumor-bone and tumor-subcutaneous microenvironments (TB- and TS-microE).. Mouse mammary tumor cells were transplanted onto the calvaria or into a subcutaneous lesion of female mice, creating a TB-microE and a TS-microE, and the mice were then treated with hOCIF. To investigate the preventive effects of hOCIF, mice were treated with hOCIF before tumor cell implantation onto the calvaria (Pre), after (Post), and both before and after (Whole). The number of CSCs and cytokine levels were evaluated by IHC and ELISA assay, respectively.. hOCIF suppressed osteolysis, and growth of mammary tumors in the TB-microE, but not in the TS-microE. In the Pre, Post, and Whole groups, hOCIF suppressed osteolysis, and cell proliferation. hOCIF increased mouse osteoprotegrin (mOPG) levels in vivo, which suppressed mammary tumor cell proliferation in vitro. These preventive effects were observed in the dose-dependent. hOCIF did not affect the induction of CSCs in either microenvironment.. While receptor activator of NF-κB ligand (RANKL) targeting therapy may not affect the induction of CSCs, RANKL is a potential target for prevention as well as treatment of breast cancer bone metastasis.

Topics: Animals; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Cytokines; Dose-Response Relationship, Drug; Female; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplastic Stem Cells; Osteolysis; Osteoprotegerin; Recombinant Proteins; Tumor Microenvironment

Aseptic loosening and menopause‑induced osteoporosis are caused by an imbalance between bone formation and osteolysis. With an aging population, the probability of simultaneous occurrence of such conditions in an elderly individual is increasing. Strontium ranelate (SR) is an anti‑osteoporosis drug that promotes bone formation and inhibits osteolysis. The present study compared the effects of SR with those of the traditional anti‑osteoporosis drug alendronate (ALN) using an ovariectomized mouse model of osteolysis. The degree of firmness of the prosthesis and the surrounding tissue was examined, a micro‑CT scan of the prosthesis and the surrounding tissue was performed, and the levels of inflammatory and osteogenic and osteoclast factors were examined. It was observed that treatment with SR and ALN improved the bond between the prosthesis and the surrounding bone tissue by reducing the degree of osteolysis, thus improving the quality of bone around the prosthesis. SR increased the secretion of osteocalcin, runt‑related transcription factor 2 and osteoprotegerin (OPG). It additionally decreased the expression of the receptor activator of nuclear factor‑κB ligand (RANKL) and consequently increased the protein ratio OPG/RANKL, whereas ALN exhibited the opposite effect. Furthermore, SR and ALN suppressed tumor necrosis factor‑α and interleukin‑1β production, with SR exerting a more marked effect. The present results demonstrate that SR and ALN may stimulate bone formation and inhibit bone resorption in the ovariectomized mouse model of wear particle‑mediated osteolysis, with SR demonstrating better effects compared with ALN.

Topics: Aged; Alendronate; Animals; Core Binding Factor Alpha 1 Subunit; Disease Models, Animal; Female; Humans; Mice; Osteocalcin; Osteoclasts; Osteogenesis; Osteolysis; Osteoporosis; Osteoprotegerin; Prosthesis Failure; Thiophenes

Topics: Animals; Bone and Bones; Bone Regeneration; Cell Differentiation; Disease Models, Animal; Eicosapentaenoic Acid; Gene Expression Profiling; Gene Expression Regulation; Inflammation; Mice; Osteoblasts; Osteoclasts; Osteogenesis; Osteolysis; Osteoprotegerin; RANK Ligand; Signal Transduction; Tumor Necrosis Factor-alpha

Wear debris-induced inhibition of bone regeneration and extensive bone resorption were common features in peri-prosthetic osteolysis (PPO). Here, we investigated the effect of melatonin on titanium particle-stimulated osteolysis in a murine calvariae model and mouse-mesenchymal-stem cells (mMSCs) culture system. Melatonin inhibited titanium particle-induced osteolysis and increased bone formation at osteolytic sites, confirmed by radiological and histomorphometric data. Furthermore, osteoclast numbers decreased dramatically in the low- and high-melatonin administration mice, as respectively, compared with the untreated animals. Melatonin alleviated titanium particle-induced depression of osteoblastic differentiation and mineralization in mMSCs. Mechanistically, melatonin was found to reduce the degradation of β-catenin, levels of which were decreased in presence of titanium particles both in vivo and in vitro. To further ensure whether the protective effect of melatonin was mediated by the Wnt/β-catenin signaling pathway, ICG-001, a selective β-catenin inhibitor, was added to the melatonin-treated groups and was found to attenuate the effect of melatonin on mMSC mineralization. We also demonstrated that melatonin modulated the balance between receptor activator of nuclear factor kappa-B ligand and osteoprotegerin via activation of Wnt/β-catenin signaling pathway. These findings strongly suggest that melatonin represents a promising candidate in the treatment of PPO.. Peri-prosthetic osteolysis, initiated by wear debris-induced inhibition of bone regeneration and extensive bone resorption, is the leading cause for implant failure and reason for revision surgery. In the current study, we demonstrated for the first time that melatonin can induce bone regeneration and reduce bone resorption at osteolytic sites caused by titanium-particle stimulation. These effects might be mediated by activating Wnt/β-catenin signaling pathway and enhancing osteogenic differentiation. Meanwhile, the ability of melatonin to modulate the balance between receptor activator of nuclear factor kappa-B ligand and osteoprotegerin mediated by Wnt/β-catenin signaling pathway, thereby suppressing osteoclastogenesis, may be implicated in the protective effects of melatonin on titanium-particle-induced bone resorption. These results suggested that melatonin can be considered as a promising therapeutic agent for the prevention and treatment of peri-prosthetic osteolysis.

Topics: Animals; beta Catenin; Bone Regeneration; Cell Differentiation; Disease Models, Animal; Intercellular Signaling Peptides and Proteins; Male; Melatonin; Mesenchymal Stem Cells; Mice, Inbred C57BL; Osteoclasts; Osteogenesis; Osteolysis; Osteoprotegerin; RANK Ligand; Skull; Titanium; Wnt Signaling Pathway

We aimed to investigate whether the RANKL/RANK/OPG system is associated with the incidence of periprosthetic osteolysis with septic loosening, and to investigate the differences of RANKL/RANK/OPG system expression in synovial fluid surrounding the normal and septic loosening hip prosthesis in canine models.. Twelve healthy adult mongrel canines were divided into two groups: experimental and control. Femoral head and stem replacements were conducted on the right side in both groups. The experimental group received the bacteria fluid intra-articular injection and the other group received the same amount of saline in the same day. The synovial fluid samples were gathered at the 1st, 2nd, 4th, 8th, 12th, 16th and 19th week after the bacteria fluid intra-articular injection for enzyme-linked immunosorbent assay (ELISA), the expression of the RANKL/RANK/OPG system.. Surgery on all animals was successful. Two dogs were excluded from the analysis of the result because of a surgery infection or death. The ELISA of the synovial fluid revealed that the ratio of RANKL/OPG showed a significant upward trend (p≤0.05) with time in the test group but the ratio of RANKL/OPG in the control group changed slowly over time (p>0.05). The ratio of RANKL/OPG value between the test and control group showed a significant upward trend, but had no statistical difference (p>0.05) over time.. It could be concluded that the RANKL/RANK/OPG system is correlated with the incidence of periprosthetic osteolysis with septic loosening. Consequently, imbalance RANKL/RANK/OPG system was related to periprosthetic osteolysis with septic loosening.

Topics: Animals; Arthroplasty, Replacement, Hip; Disease Models, Animal; Dogs; Enzyme-Linked Immunosorbent Assay; Hip Prosthesis; Osteolysis; Osteoprotegerin; Prosthesis-Related Infections; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Synovial Fluid

Aseptic loosening secondary to periprosthetic inflammatory osteolysis results from the biological response to wear particles and is a leading cause of arthroplasty failure. The origin of this inflammatory response remains unclear. We aim to validate the definite link between endoplasmic reticulum (ER) stress and particle-induced inflammatory signaling pathways in periprosthetic osteolysis. We examine the histopathologic changes of osteolysis and the expression of specific biomarkers for ER-stress-mediated inflammatory signaling pathways (IRE1α, GRP78/Bip, c-Fos, NF-κB, ROS and Ca(2+)). Moreover, pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and osteoclastogenic molecules (VEGF, OPG, RANKL and M-CSF) were assessed in clinical interface membranes and murine periosteum tissues. We found wear particles to be capable of inducing ER stress in macrophages within clinical osteolytic interface membranes and murine osteolytic periosteum tissues and to be associated with the inflammatory response and osteoclastogenesis. Blocking ER stress with sodium 4-phenylbutyrate (4-PBA) results in a dramatic amelioration of particle-induced osteolysis and a significant reduction of ER-stress intensity. Simultaneously, this ER-stress blocker also lessens inflammatory cell infiltration, diminishes the capability of osteoclastogenesis and reduces the inflammatory response by lowering IRE1α, GRP78/Bip, c-Fos, NF-κB, ROS and Ca(2+) levels. Thus, ER stress plays an important role in particle-induced inflammatory osteolysis and osteoclastogenic reactions. The pharmacological targeting of ER-stress-mediated inflammatory signaling pathways might be an appealing approach for alleviating or preventing particle-induced osteolysis in at-risk patients.

Topics: Adolescent; Aged; Animals; Cytokines; Disease Models, Animal; Disease Progression; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Female; Humans; Inflammation; Inflammation Mediators; Male; Membranes; Mice; Middle Aged; Nanoparticles; NF-kappa B; Osteoclasts; Osteolysis; Osteoprotegerin; Periosteum; Prostheses and Implants; Prosthesis Failure; Radiography; RANK Ligand; Signal Transduction; Vascular Endothelial Growth Factor A

The pathogenesis of periprosthetic osteolysis with septic loosening remains incompletely understood. The purpose of this study was to investigate whether expression of the RANKL/RANK/OPG system is altered in septic interface membranes (SIMs). Seventeen cases with a SIM, 26 cases with an aseptic interface membrane (AIM), and 12 cases with a normal synovium (NS) were assessed. Scanning and transmission electron microscopy (SEM and TEM, respectively) were used to observe the microscopic morphology of three tissue conditions. Differences in RANKL, RANK, and OPG expression at the mRNA level were assessed by real-time quantitative PCR, and differences at the protein level were assessed by immunohistochemical staining and Western blotting. SEM showed wear debris widely distributed on the AIM surface, and TEM showed Bacillus activity in the SIM. RANKL expression and the RANKL/OPG ratio were significantly increased in SIMs. Imbalance in the RANKL/RANK/OPG system is related to periprosthetic osteolysis with septic loosening but is not the only possible pathogenic mechanism.

Topics: Aged; Aged, 80 and over; Arthroplasty; Female; Humans; Joint Prosthesis; Male; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Middle Aged; Osteolysis; Osteoprotegerin; Prosthesis Failure; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; RNA, Messenger; Synovial Membrane

The relationship between bone marrow (BM) cytokine and chemokine levels, cytogenetic profiles and skeletal involvement in multiple myeloma (MM) patients is not yet defined. This study investigated a cohort of 455 patients including monoclonal gammopathy of uncertain significance (MGUS), smoldering MM and symptomatic MM patients. Skeletal surveys, positron emission tomography (PET)/computerized tomography (CT) and magnetic resonance imaging (MRI) were used to identify myeloma bone disease. Significantly higher median BM levels of both C-C motif Ligand (CCL)3 and CCL20 were found in MM patients with radiographic evidence of osteolytic lesions as compared with those without, and in all MM patients with positive PET/CT scans. BM levels of CCL3, CCL20, Activin-A and Dickkopf-1 (DKK-1) were significantly higher in patients with high bone disease as compared with patients with low bone disease. Moreover, CCL20 BM levels were significant predictors of osteolysis on X-rays by multivariate logistic analysis. On the other hand, DKK-1 levels were related to the presence of MRI lesions independently of the osteolysis at the X-rays. Our data define the relationship between bone disease and the BM cytokine and chemokine patterns highlighting the tight relationship between CCL20 BM levels and osteolysis in MM.

Topics: Aged; Bone Marrow; Chemokine CCL20; Chemokine CCL3; Chemokines; Chromosome Aberrations; Cytokines; Female; Humans; Intercellular Signaling Peptides and Proteins; Magnetic Resonance Imaging; Male; Middle Aged; Multiple Myeloma; Osteolysis; Osteoprotegerin; RANK Ligand

BACKGROUND Wear particle-induced peri-implant loosening is the most common complication affecting long-term outcomes in patients who undergo total joint arthroplasty. Wear particles and by-products from joint replacements may cause chronic local inflammation and foreign body reactions, which can in turn lead to osteolysis. Thus, inhibiting the formation and activity of osteoclasts may improve the functionality and long-term success of total joint arthroplasty. The aim of this study was to interfere with CXC chemokine receptor type 2 (CXCR2) to explore its role in wear particle-induced osteolysis. MATERIAL AND METHODS Morphological and biochemical assays were used to assess osteoclastogenesis in vivo and in vitro. CXCR2 was upregulated in osteoclast formation. RESULTS Local injection with adenovirus-mediated siRNA targeting CXCR2 inhibited titanium-induced osteolysis in a mouse calvarial model in vivo. Furthermore, siCXCR2 suppressed osteoclast formation both directly by acting on osteoclasts themselves and indirectly by altering RANKL and OPG expression in osteoblasts in vitro. CONCLUSIONS CXCR2 plays a critical role in particle-induced osteolysis, and siCXCR2 may be a novel treatment for aseptic loosening.

Topics: Adenoviridae; Animals; Bone Marrow Cells; Female; Macrophages; Mice; Mice, Inbred BALB C; Osteoclasts; Osteogenesis; Osteolysis; Osteoprotegerin; RANK Ligand; RAW 264.7 Cells; Receptors, Interleukin-8B; RNA, Small Interfering; Signal Transduction; Skull; Titanium; Up-Regulation

The detection of peptides from the calcitonin (CT) family in the periarticular tissue of loosened implants has raised hopes of opening new regenerative therapies in the process of aseptic loosening, which remains the major cause of early implant failure in endoprosthetic surgery. We have previously shown the roles of α-calcitonin gene-related peptide (α-CGRP) and the CALCA gene which encodes α-CGRP/CT in this process. To uncover the role of direct calcitonin receptor (CTR) mediated signalling, we studied particle-induced osteolysis (PIO) in a murine calvaria model with a global deletion of the CTR (CTR-KO) using μCT analysis and histomorphometry. As expected, CTR-KO mice revealed reduced bone volume compared to wild-type (WT) controls (p<0.05). In CTR-KO mice we found significantly higher RANKL (receptor activator of NF-κB ligand) expression in the particle group than in the control group. The increase in osteoclast numbers by the particles was twice as high as the increase of osteoclasts in the WT mice (400 vs. 200%). Changes in the eroded surface and actual osteolysis due to ultrahigh-molecular-weight polyethylene particles were similar in WTs and CTR-KOs. Taken together, our findings strengthen the relevance of the OPG/RANK/RANKL system in the PIO process. CTR seems to have an effect on osteoclast differentiation in this context. As there were no obvious changes of the amount of PIO in CTR deficiency, regenerative strategies in aseptic loosening of endoprosthetic implants based on peptides arising from the CT family should rather focus on the impact of α-CGRP.

Topics: Animals; Cell Count; Creatinine; Imaging, Three-Dimensional; Male; Mice, Inbred C57BL; Mice, Knockout; Organ Size; Osteocalcin; Osteoclasts; Osteolysis; Osteoprotegerin; Polyethylene; RANK Ligand; Receptors, Calcitonin; Signal Transduction; Skull; X-Ray Microtomography

Osteolytic bone damage is a major cause of morbidity in several metastatic pathologies. Current therapies using bisphosphonates provide modest improvement, but cytotoxic side effects still occur prompting the need to develop more effective therapies to target aggressive osteoclastogenesis. Increased levels of receptor activator of NF-κB ligand (TNFSF11/RANKL), leading to RANKL-RANK signaling, remain the key axis for osteoclast activation and bone resorption. Osteoprotegerin (TNFRSF11B/OPG), a decoy receptor for RANKL, is significantly decreased in patients who present with bone lesions. Despite its potential in inhibiting osteoclast activation, OPG also binds to TNF-related apoptosis-inducing ligand (TNFSF10/TRAIL), making tumor cells resistant to apoptosis. Toward uncoupling the events of TRAIL binding of OPG and to improve its utility for bone remodeling without inducing tumor resistance to apoptosis, OPG mutants were developed by structural homology modeling based on interactive domain identification and by superimposing models of OPG, TRAIL, and its receptor DR5 (TNFRSF10B) to identify regions of OPG for rational design. The OPG mutants were purified and extensively characterized for their ability to decrease osteoclast damage without affecting tumor apoptosis pathway both in vitro and in vivo, confirming their potential in bone remodeling following cancer-induced osteolytic damage.. OPG variants were developed that lack TRAIL binding, yet retain RANKL binding and suggest new possibilities for therapeutic targeting in osteolytic malignancies.

Topics: Animals; Apoptosis; Bone Neoplasms; Bone Remodeling; Cell Line, Tumor; Cell Proliferation; Humans; Male; Mice; Mice, Nude; Osteoclasts; Osteolysis; Osteoprotegerin; Prostatic Neoplasms; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand

Breast cancer is the major cause of cancer death in women worldwide. The most common site of metastasis is bone. Bone metastases obstruct the normal bone remodeling process and aberrantly enhance osteoclast-mediated bone resorption, which results in osteolytic lesions. 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is an endogenous ligand of peroxisome proliferator-activated receptor gamma (PPARγ) that has anti-inflammatory and antitumor activity at micromolar concentrations through PPARγ-dependent and/or PPARγ-independent pathways. We investigated the inhibitory activity of 15d-PGJ2 on the bone loss that is associated with breast cancer bone metastasis and estrogen deficiency caused by cancer treatment. 15d-PGJ2 dose-dependently inhibited viability, migration, invasion, and parathyroid hormone-related protein (PTHrP) production in MDA-MB-231 breast cancer cells. 15d-PGJ2 suppressed receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA levels and normalized osteoprotegerin (OPG) mRNA levels in hFOB1.19 osteoblastic cells treated with culture medium from MDA-MB-231 cells or PTHrP, which decreased the RANKL/OPG ratio. 15d-PGJ2 blocked RANKL-induced osteoclastogenesis and inhibited the formation of resorption pits by decreasing the activities of cathepsin K and matrix metalloproteinases, which are secreted by mature osteoclasts. 15d-PGJ2 exerted its effects on breast cancer and bone cells via PPARγ-independent pathways. In Balb/c nu/nu mice that received an intracardiac injection of MDA-MB-231 cells, subcutaneously injected 15d-PGJ2 substantially decreased metastatic progression, cancer cell-mediated bone destruction in femora, tibiae, and mandibles, and serum PTHrP levels. 15d-PGJ2 prevented the destruction of femoral trabecular structures in estrogen-deprived ICR mice as measured by bone morphometric parameters and serum biochemical data. Therefore, 15d-PGJ2 may be beneficial for the prevention and treatment of breast cancer-associated bone diseases.

Topics: Anilides; Animals; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Survival; Disease Models, Animal; Estrogens; Female; Humans; Male; Mice; Mice, Nude; Osteoclasts; Osteolysis; Osteoprotegerin; Ovariectomy; Parathyroid Hormone-Related Protein; PPAR gamma; Prostaglandin D2; RANK Ligand

Langerhans cell histiocytosis (LCH) is a rare disease caused by the clonal accumulation of dendritic Langerhans cells, which is often accompanied by osteolytic lesions. It has been reported that osteoclast-like cells play a major role in the pathogenic bone destruction seen in patients with LCH and these cells are postulated to originate from the fusion of DCs. However, due to the lack of reliable animal models the pathogenesis of LCH is still poorly understood. In this study, we have established a mouse model of histiocytosis- recapitulating human disease for osteolytic lesions seen in LCH patients. At 12 weeks after birth, severe bone lesions were observed in our multisystem histiocytosis (Mushi) model, when CD8α conventional dendritic cells (DCs) are transformed (MuTuDC) and accumulate. Most importantly, our study demonstrates that bone loss in LCH can be accounted for the transdifferentiation of MuTuDCs into functional osteoclasts both in vivo and in vitro. Moreover, we have shown that injected MuTuDCs reverse the osteopetrotic phenotype of oc/oc mice in vivo. In conclusion, our results support a crucial role of DCs in bone lesions in histiocytosis patients. Furthermore, our new model of LCH based on adoptive transfer of MuTuDC lines, leading to bone lesions within 1-2 weeks, will be an important tool for investigating the pathophysiology of this disease and ultimately for evaluating the potential of anti-resorptive drugs for the treatment of bone lesions.

Topics: Animals; Bone and Bones; Bone Density Conservation Agents; Cell Line; Cell Transdifferentiation; Dendritic Cells; Diphosphonates; Disease Models, Animal; Histiocytosis, Langerhans-Cell; Humans; Langerhans Cells; Mice; Mice, Inbred C57BL; Mice, Transgenic; Osteoclasts; Osteolysis; Osteoprotegerin

Periprosthetic osteolysis, involving RANK/RANKL/osteoprotegerin (OPG) and TNF-α/NFκB signaling, contributes to bone resorption and inflammation. We constructed lentivirus vectors to inhibit TNF-α and enhance OPG expression and assessed their impacts on wear debris-induced inflammation and osteoclastogenesis in an osteoclast/osteoblast coculture system.. We transduced mouse osteoblastic MC3T3-E1 cells with Lenti-negative control (Lenti-NC), Lenti-OPG or Lenti-siTNFα-OPG, and murine macrophage/monocyte RAW264.7 cells with Lenti-NC, Lenti-TNF-α siRNA or Lenti-siTNFα-OPG. Then, TNF-α and OPG protein levels were evaluated by enzyme-linked immunosorbent assay. We cocultured transduced MC3T3-E1 and RAW264.7 cells in transwell chambers in the presence of 0.1 mg/mL Ti particles to investigate the capacity of TNF-α inhibition to reduce wear debris-induced inflammation. We also assessed mRNA levels TNF-α, IL-1β, IL-6 and OPG by RT-PCR as well as osteoclastogenesis by tartrate-resistant acid phosphatase.. Lenti-siTNFα-OPG ameliorated Ti-particle-induced expression of TNF-α, IL-1β, IL-6 in MC3T3-E1/RAW264.7 cocultures, while enhancing mRNA and protein levels of OPG, and reducing the fraction of tartrate-resistant acid phosphatase (TRAP)+ cells.. Lenti-siTNFα-OPG can inhibit the wear debris-induced inflammatory responses and osteoclastogenesis in vitro, and may represent a promising therapeutic candidate for the treatment or prevention of wear particle-induced osteolysis.

Topics: Animals; Cytokines; Inflammation; Mice; Osteoclasts; Osteolysis; Osteoprotegerin; Tumor Necrosis Factor-alpha

Periprosthetic osteolysis is the leading reason for THA revision. The relationship of serum biomarkers with severe radiographic periprosthetic osteolysis has not been defined but may be important to direct future research and clinical therapeutics.. We determined whether there was an association between measurable inflammatory markers (high-sensitivity C-reactive protein [hsCRP]) or inflammatory mediators (tumor necrosis factor α [TNF-α], IL-1β, IL-6, receptor activator of nuclear factor κB ligand [RANKL], and osteoprotegerin [OPG]) and periprosthetic osteolysis.. We identified 15 patients with THAs scheduled for revision surgery because of severe periprosthetic osteolysis. For each study patient, a nonosteolytic, pain-free control patient with THAs was identified and matched for age, sex, time since initial THA, acetabular and femoral component prosthesis material, and prosthesis wear within 1.0 mm/year using a manual wear analysis technique. Overall, the study and control patients had a mean wear rate of 0.25 mm/year since index THA. There were no differences in baseline characteristics between study and control patients in age, sex, BMI, Charlson Comorbidity Index, time since initial THA, UCLA activity score, and acetabular and femoral component type. Serum hsCRP, IL-1β, IL-6, TNF-α, RANKL, and OPG were measured by ELISA in duplicate assays. Differences in values were assessed using the Wilcoxon rank-sum test.. Median TNF-α levels were higher in study patients than in controls (7.1 pg/mL [SD, 11.6 pg/mL] versus 1.5 pg/mL [SD, 1.3 pg/mL]) (p < 0.01). Median IL-6 levels tended to be higher in study patients than in controls (8.9 pg/mL [SD, 13.2 pg/mL] versus 3.5 pg/mL [SD, 0.7 pg/mL]) (p = 0.09). The other serum inflammatory proteins and mediators of bone turnover were not different between groups.. TNF-α is elevated in patients with osteolysis compared to matched controls. The role of TNF-α and its potential as a target of nonsurgical therapy to prevent osteolysis warrant further investigation in larger, prospective studies.

Topics: Aged; Aged, 80 and over; Arthroplasty, Replacement, Hip; Biomechanical Phenomena; C-Reactive Protein; Case-Control Studies; Chi-Square Distribution; Enzyme-Linked Immunosorbent Assay; Female; Hip Joint; Hip Prosthesis; Humans; Inflammation Mediators; Interleukin-1beta; Interleukin-6; Male; Middle Aged; Osteoarthritis, Hip; Osteolysis; Osteoprotegerin; Prosthesis Design; Prosthesis Failure; Radiography; RANK Ligand; Reoperation; Risk Factors; Stress, Mechanical; Treatment Outcome; Tumor Necrosis Factor-alpha; Up-Regulation

Osteosarcoma is the most common primary malignant tumor of bone and accounts for around 50% of all primary skeletal malignancies. In addition to novel chemotherapies, there is a need for adjuvant therapies designed to inhibit osteosarcoma proliferation and tumor-induced osteolysis to attenuate tumor expansion and metastasis. As such, studies on the efficacy of bisphosphonates on human osteosarcoma are planned after feasibility studies determined that the bisphosphonate zoledronic acid (ZOL) can be safely combined with conventional chemotherapy. However, the molecular mechanisms responsible for, and means of inhibiting, osteosarcoma-induced osteolysis are largely unknown. We establish that osteosarcoma growth directly correlates with tumor-induced osteolysis and activation of osteoclasts in vivo. In vitro, tumor cells were determined to expresses surface, but not soluble, receptor activator of NF-κB ligand (RANKL) and stimulated osteoclastogenesis in a manner directly proportional to their malignant potential. In addition, an aggressive osteosarcoma cell line was shown to secrete monocyte chemoattractant protein-1 (MCP-1), resulting in robust monocyte migration. Because MCP-1 is a key cytokine for monocyte recruitment and surface-bound RANKL strongly supports local osteoclastogenesis, we suggest that high levels of these signaling molecules are associated with the aggressive potential of osteosarcoma. Consistent with these findings, abundant expression of RANKL/MCP-1 was observed in tumor in vivo, and MCP-1 plasma levels strongly correlated with tumor progression and osteolysis. ZOL administration directly attenuates osteosarcoma production of RANKL/MCP-1, reducing tumor-induced bone destruction. In vivo, these findings also correlated with significant reduction in osteosarcoma growth. ZOL attenuates tumor-induced osteolysis, not only through direct inhibition of osteoclasts, but also through direct actions on tumor expression of osteoclast activators. These data provide insight regarding the effect of ZOL on osteosarcoma essential for designing the planned upcoming prospective randomized trials to determine the efficacy of bisphosphonates on osteosarcoma in humans.

Topics: Animals; Bone Resorption; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chemokine CCL2; Diphosphonates; Humans; Imidazoles; Mice; Monocytes; Neoplasm Invasiveness; Osteoclasts; Osteogenesis; Osteolysis; Osteoprotegerin; Osteosarcoma; RANK Ligand; Zoledronic Acid

Bone metastasis is a serious complication in patients with lung cancer, occurring in up to 40% of patients. Tumor cell-mediated osteolysis occurs ultimately through induction of RANK ligand (RANKL) within the bone stroma although this hypothesis has not been tested extensively in the setting of non-small-cell lung cancer (NSCLC). By using two novel NSCLC bone metastasis mouse models, we examined the effects of RANKL inhibition on osteolysis and tumor progression.. We treated mice bearing skeletal NSCLC tumors with osteoprotegerin-Fc (OPG-Fc) to assess whether osteoclast inhibition through RANKL inhibition would affect bone metastases at early or late stages of bone colonization. Progression of skeletal tumor was determined by radiography, longitudinal bioluminescent imaging, and histological analyses.. OPG-Fc reduced development and progression of radiographically evident osteolytic lesions and also significantly reduced skeletal tumor progression in both NSCLC bone metastasis models. In the H1299 human NSCLC bone metastasis model, OPG-Fc plus docetaxel in combination resulted in significantly greater inhibition of skeletal tumor growth compared with either single agent alone. The observed ability of RANKL inhibition to reduce NSCLC osteolytic bone destruction or skeletal tumor burden was associated with decreases in tumor-associated osteoclasts.. These results demonstrate that RANKL is required for the development of tumor-induced osteolytic bone destruction caused by NSCLC cells in vivo. RANKL inhibition also reduced skeletal tumor burden, presumably through the indirect mechanism of blocking tumor-induced osteoclastogenesis and resultant production of growth factors and calcium from the bone microenvironment. RANKL inhibition also provided an additive benefit to docetaxel treatment by augmenting the reduction of tumor burden.

Topics: Animals; Anticoagulants; Bone Neoplasms; Carcinoma, Non-Small-Cell Lung; Enoxaparin; Female; Humans; Lung Neoplasms; Mice; Mice, Nude; Osteolysis; Osteoprotegerin; RANK Ligand; Survival Rate; Tumor Burden; Tumor Cells, Cultured

Recently, it was found that α-Calcitonin gene-related peptide (CGRP) was associated with breast cancer metastases, but the role of CGRP in interaction between breast cancer and osteoblast during bone metastases is not clear. Here, we investigated the effect of CGRP on osteoblast in co-culture system with breast cancer. Using a breast cancer-osteoblast co-culture system, we chose MDA-MB-231 for breast cancer and human cell line MG-63 for osteoblast. CGRP was added to this co-culture system. The expression levels of the Runx2, RANK1, and osteoprotegerin (OPG) were analyzed using real-time PCR and western blot. CGRP receptors were investigated by immunofluorescence. We found that breast cancer cells cause osteolysis lesions by upregulating Runx2 expression, decreasing OPG expression, and increasing RANKL expression in osteoblasts. Our data prove that CGRP can regulate osteoclast coupling genes in osteoblast by increasing OPG, and decreasing RANKL and Runx2 expressions in a time-dependent manner; and inhibit those osteolytic factors induced by interaction between breast cancer cells and osteoblast. This inhibition could be abolished by the CGRP antagonist, CGRP8-37. In conclusion, calcitonin receptor-like receptor is the key player for CGRP's effect in this co-culture system.

Topics: Bone Neoplasms; Breast Neoplasms; Calcitonin Gene-Related Peptide; Cell Line, Tumor; Coculture Techniques; Gene Expression Regulation; Humans; Osteoblasts; Osteolysis; Osteoprotegerin; Peptide Fragments; RANK Ligand

Osteoprotegerin (OPG) is a secreted member of the TNF receptor superfamily, which binds to the receptor activator of nuclear factor κB ligand (RANKL) and inhibits osteoclast activity and bone resorption. Systemic administration of recombinant OPG was previously shown to inhibit tumor growth in bone and to prevent cancer-induced osteolysis. In this study, we examined the effect of OPG, when produced locally by breast cancer cells located within bone, using a mouse model of osteolytic breast cancer. MDA-MB-231-TXSA breast cancer cells, tagged with a luciferase reporter gene construct and engineered to overexpress full-length human OPG, were transplanted directly into the tibial marrow cavity of nude mice. Overexpression of OPG by breast cancer cells protected the bone from breast cancer-induced osteolysis and diminished intra-osseous tumor growth but had no effect on extra-skeletal tumor growth. This effect was associated with a significant reduction in the number of osteoclasts that lined the bone surface, resulting in a net increase in bone volume. Despite limiting breast cancer-mediated bone loss, OPG overexpression resulted in a significant increase in the incidence of pulmonary metastasis. Our results demonstrate that inhibition of osteoclastic bone resorption by OPG when secreted locally by tumors in bone may affect the behaviour of cancer cells within the bone microenvironment and their likelihood of spreading and establishing metastasis elsewhere in the body.

Topics: Animals; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Humans; Mice; Mice, Nude; Neoplasm Invasiveness; Osteolysis; Osteoprotegerin; Soft Tissue Neoplasms; Transfection; X-Ray Microtomography

Wear-particle-induced osteolysis is considered to be the main reason for revision after arthroplasty. Although the exact mechanism remains unclear, inflammatory osteoclastogenesis plays an important role in this process. Strontium ranelate (SR) was found to have a therapeutic effect on osteoporosis in postmenopausal women. Based on prior studies, the present authors hypothesized that SR prevents wear-particle-induced osteolysis through restraining inflammatory osteoclastogenesis. The present study used 80 male C57BL/J6 mice to test this hypothesis in a murine osteolysis model. All experimental animals were randomly divided into four groups: a control group; a SR group; a titanium group; and a titanium+SR group. Once titanium particles had been implanted in mice, the mice were administered SR (900 mg kg(-1) day(-1)) by gavage for 14 days. After 14 days, the calvaria were collected for micro-computed tomography (μCT), histological and molecular analysis. The results of μCT and histomorphometric analysis demonstrated that SR markedly inhibited bone resorption and the generation of tartrate-resistant acid-phosphatase-positive cells in vivo, compared with titanium-stimulated calvaria. Reverse transcription polymerase chain reaction and ELISAs showed that SR stimulated the mRNA and protein expression of osteoprotegerin, and inhibited gene and protein expression of receptor activators of nuclear factor-kappa B ligand in titanium-particle-charged calvaria. In addition, SR obviously reduced the secretion of tumor necrosis factor-α and interleukin-1β in the calvaria of the titanium group. It was concluded that SR inhibits titanium-induced osteolysis by restraining inflammatory osteoclastogenesis, and that it could be developed as a new drug to prevent and treat aseptic loosening.

Topics: Acid Phosphatase; Animals; Inflammation; Interleukin-1beta; Isoenzymes; Male; Mice, Inbred C57BL; Osteoclasts; Osteogenesis; Osteolysis; Osteoprotegerin; RANK Ligand; RNA, Messenger; Skull; Staining and Labeling; Tartrate-Resistant Acid Phosphatase; Thiophenes; Titanium; Tumor Necrosis Factor-alpha; X-Ray Microtomography

To investigate the expression changes of the receptor activator of nuclear factor-κB ligand (RANKL) in the peripheral blood of patients with aseptic loosening of the implant after total hip arthroplasty (THA) by comparing with that of patients with femoral neck fracture and to analyze the correlation between RANKL expression and aseptic loosening.. Between January 2008 and January 2013, the peripheral blood were harvested from 58 patients with aseptic loosening of the implant after THA (trial group) and 63 patients with femoral neck fracture (control group). The 2 groups were well matched, with no significant differences in age and gender (P > 0.05). The expressions of the RANKL mRNA and RANKL protein were evaluated by quantitative real-time PCR and Western blot respectively. At the same time, the concentration of RANKL was also measured by ELISA.. The expression of the RANKL mRNA in the trial group was 18.30 ± 1.09, which was significantly higher than that of control group (1.00 ± 0.05) (t = 125.390, P = 0.000). The relative RANKL protein expression values in trial group and control group were 0.856 ± 0.254 and 0.404 ± 0.102 respectively, showing significant difference (t = 13.032, P = 0.000). The results of ELISA showed that the concentration of RANKL in trial group [(3.553 5 ± 0.129 7) ng/mL] was significantly higher than that of control group [(1.912 3 ± 0.126 2) ng/ mL] (t = 18.124, P = 0.000).. The high RANKL expression in peripheral blood is probably correlated with aseptic loosening of the implant in patients undergoing THA, which possibly is the prognostic factor of aseptic loosening of the implant.

Topics: Arthroplasty, Replacement, Hip; Blotting, Western; Hip Prosthesis; Humans; Osteolysis; Osteoprotegerin; Prosthesis Failure; Prosthesis-Related Infections; RANK Ligand; Real-Time Polymerase Chain Reaction; Receptor Activator of Nuclear Factor-kappa B

Osteolytic bone diseases are a prominent feature of multiple myeloma (MM), resulting from aberrant osteoclastic bone resorption that is uncoupled from osteoblastic bone formation. Myeloma stimulates osteoclastogenesis, which is largely dependent on an increase in receptor activator of NF-κB ligand (RANKL) and a decrease in osteoprotegerin (OPG) within the bone marrow milieu. Recently, brain-derived neurotrophic factor (BDNF) was identified as a MM-derived factor that correlates with increased RANKL levels and contributes to osteolytic bone destruction in myeloma patients. Because tyrosine receptor kinase B (TrkB), the receptor of BDNF, is abundantly expressed in osteoblasts, we sought to evaluate the role of BDNF/TrkB in myeloma-osteoblast interactions and the effect of this pathway on the RANKL/OPG ratio and osteoclastogenesis. Coculture systems constructed with noncontact transwells revealed that, in vitro, MM-derived BDNF increased RANKL and decreased OPG production in osteoblasts in a time- and dose-dependent manner. These effects were completely abolished by a specific small interfering RNA for TrkB. BDNF regulates RANKL/OPG expression in osteoblasts through the TrkB/ERK pathway. To investigate the biological effects of BDNF on myeloma in vivo, a SCID-RPMI8226 mice model was constructed using lentiviral short hairpin RNA-transfected RPMI8226 cells. In this system, stable knockdown of BDNF in MM cells significantly restored the RANKL/OPG homostasis, inhibited osteolytic bone destruction and reduced angiogenesis and tumor burden. Our studies provide further support for the potential osteoclastogenic effects of BDNF, which mediates stroma-myeloma interactions to disrupt the balance of RANKL/OPG expression, ultimately increasing osteoclastogenesis in MM.

Topics: Animals; Brain-Derived Neurotrophic Factor; Cell Line, Tumor; Female; Gene Silencing; Humans; MAP Kinase Signaling System; Mice; Multiple Myeloma; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor, trkB; RNA, Small Interfering; Tumor Burden

Our aim was to define the effect of multiple biomarkers of osteolysis or bone remodelling in the early detection of aseptic loosening (AL) of total hip arthroplasty (THA).. One hundred subjects were recruited, including 31 candidates for revision THA (Late AL group), 15 patients who had undergone THA and had clinical and radiographic evidence of AL (early AL group), 19 patients with no sign of AL (stable group), and 40 healthy volunteers. Plasma levels of osteoprotegerin (OPG), receptor activator of nuclear factor-kappaB ligand (RANKL), cross-linked N-terminal telopeptide (NTX), procollagen I C-terminal extension peptide (PICP), tumour necrosis factor-alpha (TNF-α), and interleukin (IL)-1β1 were measured using an immunoenzymatic method. The outcomes of biomarkers were analysed separately and synthetically using Revman software.. The plasma level of OPG, RANKL, NTX, TNF-α, and IL-1β declined from late AL, early AL, stable to the healthy group, while the level of PICP inclined reversely. There was a significant difference in synthetic analysis of six biomarkers between the AL group and the stable group, and between the stable group and the healthy group (both p = 0.02). Heterogeneity of six biomarkers in either comparison was extremely low (both I(2) =0). Patients who had cemented implants had significantly higher levels of TNF-α than patients with cementless varieties (p = 0.042).. There was significant change in the plasma level of multiple biomarkers in patients with prosthetic AL of THA, especially in the cemented arthroplasties and in patients without traditional clinical or radiographic evidence of AL.

Topics: Adult; Aged; Aged, 80 and over; Arthroplasty, Replacement, Hip; Biomarkers; Bone Remodeling; Case-Control Studies; Collagen Type I; Early Diagnosis; Female; Femur Head Necrosis; Hip Dislocation; Hip Prosthesis; Humans; Interleukin-1beta; Male; Middle Aged; Osteoarthritis, Hip; Osteolysis; Osteoprotegerin; Peptide Fragments; Peptides; Procollagen; Prosthesis Failure; RANK Ligand; Tumor Necrosis Factor-alpha

To determine the mechanism of neuroblastoma (NB) bone invasion/metastasis, it is necessary to investigate the bone invasion/metastasis-related factors in the bone invasion/metastasis process. Some evidence has suggested that various proteins were involved in bone osteolytic response. The invasion/metastasis property and gene expression of NB, however, are still unknown.. Single-cell suspensions of SY5Y and KCNR cells were injected directly into the femur of nude mice. Radiological and histological analyses, immunohistochemistry analyses, and western blot assay were performed to characterize bone metastasis mechanism in these bone metastasis models.. SY5Y and KCNR NB cells result in osteolytic responses in bone metastasis model. Osteoprotegerin (OPG), receptor activator of NF-kappaB ligand (RANKL), parathyroid hormone-related peptide (PTHrP), endothelin 1 (ET-1), and CXCR4 were examined and compared among in vitro, in vivo, and normal bone, respectively. PTHrP, OPG, RANKL, and ET-1 except CXCR4 in SY5Y and KCNR NB cells xenografts were strikingly upregulated compared with normal bone and NB cells. However, significantly stronger expression of PTHrP and RANKL was presented than ET-1 and OPG; furthermore, the ratios of expression of PTHrP, RANKL to OPG, and ET-1 were also markedly increased in vivo versus in vitro.. Our study provided evidence that NB cell may enhance bone invasion through PTHrP, OPG, RANKL, and ET-1, especially PTHrP and RANKL which may display stronger effects. CXCR4 appeared not participating in bone invasion, but in tumor growth, and homing to bone. Targeting PTHrP, OPG, ET-1, and RANKL may provide a new insight and method for patient therapy by inhibiting NB bone metastasis and invasiveness.

Topics: Animals; Blotting, Western; Bone Neoplasms; Cell Line, Tumor; Endothelin-1; Femur; Humans; Immunohistochemistry; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Neuroblastoma; Osteolysis; Osteoprotegerin; Parathyroid Hormone-Related Protein; RANK Ligand; Receptors, CXCR4

Giant cell tumor (GCT) of bone is a benign but locally aggressive neoplasm of bone. However, molecular mechanisms underlying osteolysis in GCT have not been deeply understood. The aim of this study was to investigate one of the possible mechanisms underlying the up-regulation of receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG) expression. First, we performed an immunohistochemical study on transforming growth factor-β1 (TGF-β1) expression in 83 cases with GCT and found that increased TGF-β1 staining was significantly correlated with Campanacci stages(Spearman's correlation = 0.335, p = 0.002). Next, we investigated the mechanism of the effect of TGF-β1 on osteolysis of GCT and examined the effects of TGF-β1 plus or minus specific inhibitor of Smad3 (SIS3) on the expression of RANKL/OPG ratio at the mRNA and protein levels in two primary GCT cell lines. The results clearly indicated that TGF-β1 is capable of significantly increasing RANKL/OPG ratio (p GCT1 = 0.000, p GCT2 = 0.000) and that SIS3 is capable of reversing the ratio, suggesting that Smad3 is the key to TGF-β1-induced increased the ratio. In the co-culture system, we found that SIS3 reversed the effects of TGF-β1-induced osteoclast formation in the co-culture system (p GCT1 = 0.000, p GCT2 = 0.000). Our findings indicate that TGF-β1 plays an important role in the osteolysis of GCT via Smad3.

Topics: Adult; Bone Neoplasms; Cell Differentiation; Cell Line, Tumor; Coculture Techniques; Female; Giant Cell Tumor of Bone; Humans; Male; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; RNA, Messenger; Smad3 Protein; Transforming Growth Factor beta1; Up-Regulation; Young Adult

Wear particle-induced vascularized granulomatous inflammation and subsequent inflammatory osteolysis is the most common cause of aseptic loosening after total joint replacement (TJR); however, the precise mechanism by which this occurs is unclear. This study investigates the effects of the proteasome inhibitor bortezomib (Bzb) on the expression of key biochemical markers of bone metabolism and vascularised granulomatous tissues, such as receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), vascular endothelial growth factor (VEGF) and tumor necrosis factor receptor-associated factor 6 (TRAF6). In addition, the effect of Bzb on apoptosis of CD68+ cells was examined. A total of 32 female BALB/C mice were randomly divided into four groups. After implantation of calvaria bone from syngeneic littermates, titanium (Ti) particles were injected into established air pouches for all mice (excluding negative controls) to provoke inflammatory osteolysis. Subsequently, Bzb was administered at a ratio of 0, 0.1, or 0.5 mg/kg on day 1, 4, 8, and 11 post-surgery to alleviate this response. All of the air pouches were harvested 14 days after the surgical procedure and were processed for molecular and histological analysis. The results demonstrated that Ti injection elevated the expression of RANKL, OPG, VEGF, and TRAF6 at both the gene and protein levels, increased counts of infiltrated cells and thickness of air pouch membranes, and elevated the apoptosis index (AI) of CD68+ cells. Bzb treatment significantly improved Ti particle-induced implanted bone osteolysis, attenuated vascularised granulomatous tissues and elevated AI of CD68+ cells. Therefore, the proteasome pathway may represent an effective therapeutic target for the prevention and treatment of aseptic loosening.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Apoptosis; Arthroplasty, Replacement; Boronic Acids; Bortezomib; Female; Inflammation; Mice; Mice, Inbred BALB C; Osteolysis; Osteoprotegerin; Prosthesis Failure; Proteasome Inhibitors; Pyrazines; Random Allocation; RANK Ligand; Titanium; TNF Receptor-Associated Factor 6; Vascular Endothelial Growth Factors

Osteoclast formation is central to bone metabolism, occurring when myelomonocytic progenitors are stimulated by membrane-bound receptor activator of NFκB ligand (RANKL) on osteoblasts. Osteolytic hormones induce osteoblast RANKL expression, and reduce production of RANKL decoy receptor osteoprotegerin (OPG). However, rather than RANKL and OPG mRNA or protein levels, to measure hormonally-induced osteoclastogenic stimuli the net RANKL activity at the osteoblast surface needs to be determined. To estimate this we developed a cell reporter approach employing pre-osteoclast RAW264.7 cells transfected with luciferase reporter constructs controlled by NFκB (NFκB-RAW) or NFATc1 (NFAT-RAW)-binding promoter elements. Strong signals were induced in these cells by recombinant RANKL over 24h. When NFκB-RAW cells were co-cultured on osteoblastic cells (primary osteoblasts or Kusa O cells) stimulated by osteolytic factors 1,25(OH)(2) vitamin D(3) (1,25(OH)(2)D(3)) and prostaglandin E(2) (PGE(2)), a strong dose dependent signal in NFκB-RAW cells was induced. These signals were completely blocked by soluble recombinant RANKL receptor, RANK.Fc. This osteoblastic RANKL activity was sustained for 3 days in Kusa O cells; with 1,25(OH)(2)D(3) withdrawal, RANKL-induced signal was still detectable 24 h later. However, conditioned medium from stimulated osteoblasts induced no signal. TGFβ treatment inhibited osteoclast formation supported by 1,25(OH)(2)D(3)-treated Kusa O cells, and likewise blocked RANKL-dependent signals in NFAT-RAW co-cultured with these cells. These data indicate net RANKL stimulus at the osteoblast surface is increased by 1,25(OH)(2)D(3) and PGE(2), and suppressed by TGFβ, in line with their effects on RANKL mRNA levels. These results demonstrate the utility of this simple co-culture-based reporter assay for osteoblast RANKL activity.

Topics: Animals; Biological Assay; Calcitriol; Cell Line; Cell Membrane; Coculture Techniques; Dinoprostone; Genes, Reporter; Luciferases; Mice; Mice, Inbred C57BL; NF-kappa B; Osteoblasts; Osteolysis; Osteoprotegerin; Promoter Regions, Genetic; RANK Ligand; Transforming Growth Factor alpha

Icariin has been reported to enhance bone healing and treat osteoporosis. In this study, we examined the detail molecular mechanisms of icariin on lipopolysaccharide (LPS)-induced osteolysis. Our hypothesis is that icariin can inhibit osteoclast differentiation and bone resorption by suppressing MAPKs/NF-κB regulated HIF-1α and PGE(2) synthesis. After treatment with icariin, the activity of osteoclasts differentiation maker, tatrate resistances acid phosphatease (TRAP), significantly decreased at the concentration of 10(-8)M. Icariin (10(-8)M) reduced the size of LPS-induced osteoclasts formation, and diminished their TRAP and acid phosphatease (ACP) activity without inhibition of cell viability. Icariin also inhibited LPS-induced bone resorption and interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) expression. The gene expression of osteoprotegerin (OPG) was up-regulated, while receptor activator of NF-κB ligand (RANKL) was down-regulated. Icariin also inhibited the synthesis of cyclo-oxygenase type-2 (COX-2) and prostaglandin E(2) (PGE(2)). In addition, icariin had a dominant repression effect on LPS-induced hypoxia inducible factor-1α (HIF-1α) expression of osteoclasts. On osteoclasts, icariin suppresses LPS-mediated activation of the p38 and JNK; while on the osteoblasts, icariin reduced the LPS-induced activation of ERK1/2 and I-kappa-B-alpha (IκBα), but increased the activation of p38. In conclusion, we demonstrated that icariin has an in vitro inhibitory effects on osteoclasts differentiation that can prevent inflammatory bone loss. Icariin inhibited LPS-induced osteoclastogenesis program by suppressing activation of the p38 and JNK pathway.

Topics: Animals; Bone Resorption; Cell Differentiation; Cyclooxygenase 2; Dinoprostone; Epimedium; Female; Flavonoids; Gene Expression; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation Mediators; Interleukin-6; Lipopolysaccharides; Mice; Mice, Inbred ICR; Mitogen-Activated Protein Kinases; NF-kappa B; Osteoclasts; Osteolysis; Osteoporosis; Osteoprotegerin; Plant Extracts; RANK Ligand; RNA, Messenger; Tumor Necrosis Factor-alpha

The aim of this study was to evaluate the immunohistochemical expression of molecules involved in osteoclastogenesis, including the receptor activator of nuclear factor kappa B (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) in odontogenic keratocysts (OKCs), which has been named as a keratocystic odontogenic tumour by the WHO, and compare their expression with radicular cysts and ameloblastomas. RANK is a member of tumour necrosis factor receptor family and it is activated by RANK ligand. OPG binds to RANKL and inactivates it. The imbalance of these factors could cause the differential bone resorption activity in some diseases and tumours. The expression of these molecules was evaluated in ameloblastomas (n = 20), OKCs (n = 20), and radicular cysts (n = 20) by immunohistochemistry. Immunohistochemical reactivity for RANK, RANKL, and OPG was detected in neoplastic and nonneoplastic epithelium and connective tissue cells. RANK showed the greatest expression in OKCs followed by ameloblastomas, with the lowest expression seen in radicular cysts. Expression of RANKL was detected in all lesions and no significant differences were observed between groups. OPG was expressed very low in all groups. In the stroma, the number of RANK positive cells was higher in OKCs when compared with ameloblastomas and radicular cysts but radicular cyst had higher numbers of RANKL positive cells in the stroma than ameloblastomas. The molecular system of RANK/RANKL/OPG is variably expressed in OKCs, radicular cysts, and ameloblastomas and this system may be involved in the osteoclastogenic mechanisms in OKCs and ameloblastomas. Advanced studies could further clarify the role of RANK, RANKL, and OPG in mediating tumour associated bone osteolysis.

Topics: Adult; Ameloblastoma; Female; Humans; Immunohistochemistry; Jaw Neoplasms; Male; Middle Aged; Odontogenic Cysts; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction

Postmenopausal osteoporosis may modulate bone response to wear debris. In this article, we evaluate the influence of oestrogen deficiency on experimental particle-induced osteolysis.. Polyethylene (PE) particles were implanted onto the calvaria of normal controls, sham-ovariectomized (OVX), OVX mice and OVX mice supplemented with oestrogen (OVX+E). After 14 days, seven skulls per group were analyzed using a high-resolution micro-computed tomography (micro-CT) and histomorphometry, and for tartrate-specific alkaline phosphatase. Five calvariae per group were cultured for the assay of IL-1β, IL-6, TNF-α and receptor activator of the nuclear factor κB (RANKL) secretion using quantitative ELISA. Serum IL-6 concentrations were obtained. The expression of RANKL and osteoprotegerin (OPG) mRNA were evaluated using real-time PCR.. As assessed by μCT and by histomorphometry, PE particles induced extensive bone resorption and an intense inflammatory reaction in normal controls, sham-OVX and OVX+E mice, but not in the OVX mice group. In normal controls, sham-OVX and OVX+E mice, PE particles induced an increase in serum IL-6, in TNF-α and RANKL local concentrations, and resulted in a significant increase in RANKL/OPG messenger RNA (mRNA) ratio. Conversely, these parameters remained unchanged in OVX mice after PE implantation.. Oestrogen privation in the osteolysis murine model ultimately attenuated osteolytic response to PE particles, suggesting a protective effect. This paradoxical phenomenon was associated with a down-regulation of pro-resorptive cytokines. It is hypothesized that excessive inflammatory response was controlled, illustrated by the absence of increase of serum IL-6 in OVX mice after PE implantation.

Topics: Animals; Disease Models, Animal; Estrogens; Female; Gene Expression; Interleukin-1beta; Interleukin-6; Mice; Mice, Inbred C57BL; Organ Culture Techniques; Osteolysis; Osteoporosis; Osteoprotegerin; Ovariectomy; Polyethylene; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Skull; Tumor Necrosis Factor-alpha; X-Ray Microtomography

Periprosthetic osteolysis is a major cause of aseptic loosening in joint arthroplasty. This study investigates the impact of CT (calcitonin) deficiency and CT substitution under in-vivo circumstances on particle-induced osteolysis in Calca -/- mice.. We used the murine calvarial osteolysis model based on ultra-high molecular weight polyethylene (UHMWPE) particles in 10 C57BL/6J wild-type (WT) mice and twenty Calca -/- mice. The mice were divided into six groups: WT without UHMWPE particles (Group 1), WT with UHMWPE particles (Group 2), Calca -/- mice without UHMWPE particles (Group 3), Calca -/- mice with UHMWPE particles (Group 4), Calca -/- mice without UHMWPE particles and calcitonin substitution (Group 5), and Calca -/- mice with UHMWPE particle implantation and calcitonin substitution (Group 6). Analytes were extracted from serum and urine. Bone resorption was measured by bone histomorphometry. The number of osteoclasts was determined by counting the tartrate-resistant acid phosphatase (TRACP) + cells.. Bone resorption was significantly increased in Calca -/- mice compared with their corresponding WT. The eroded surface in Calca -/- mice with particle implantation was reduced by 20.6% after CT substitution. Osteoclast numbers were significantly increased in Calca -/- mice after particle implantation. Serum OPG (osteoprotegerin) increased significantly after CT substitution.. As anticipated, Calca -/- mice show extensive osteolysis compared with wild-type mice, and CT substitution reduces particle-induced osteolysis.

Topics: Animals; Bone Density Conservation Agents; Bone Resorption; Calcitonin; Cell Count; Disease Models, Animal; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microspheres; Osteoclasts; Osteolysis; Osteoprotegerin; Polyethylene; Prosthesis Failure; Skull; Treatment Outcome

To test the effect of recombinant interleukin-4 (IL-4) and recombinant osteoprotegerin (OPG) in suppressing bone resorption induced by polyethylene wear particles... A cranial bone allograft was introduced into the air pouches induced on the back of BALB/c mice, followed by injection of 1 ml suspension of polyethylene particles into the pouches. The mouse models were then divided into 3 groups to receive injections of saline (control), IL-4 alone, or IL-4 and OPG into the pouches. The tissues were harvested 21 days after bone implantation for molecular and histological analyses.. Polyethylene wear particles-stimulated inflammatory responses (increased cellular infiltration and IL-1 and TNF production) were markedly reduced by IL-4 treatment either alone or combined with OPG (P<0.05). Polyethylene particles significantly increased tartrate-resistant acid phosphatase (TRAP) staining and bone absorption of the implanted bone graft, and IL-4 treatment, either alone or combined with OPG, obviously reduced the osteolysis induced by polyethylene particles (P<0.05).. IL-4 offers protection against polyethylene wear debris-induced inflammation and bone resorption in this mouse model. IL-4 combined with OPG can be a feasible and effective therapeutic approach to the treatment and prevention of polyethylene wear debris-associated osteolysis and aseptic loosening of the prosthetic components.

Topics: Animals; Bone Resorption; Disease Models, Animal; Female; Interleukin-4; Mice; Mice, Inbred BALB C; Orthopedic Fixation Devices; Osteolysis; Osteoprotegerin; Polyethylene; Recombinant Proteins

The development and spread of tumors is associated with the ability of malignant cells to avoid detection and subsequent elimination by the immune system, to grow in non-native sites and to avoid programmed or induced cell death. In addition to the well-described role of osteoprotegerin in the regulation of bone turnover, there is an emerging evidence that osteoprotegerin may have an additional function due to its ability to bind and inhibit the members of the tumor necrosis factor (TNF)-superfamily, such as TNF-alpha and TNF-related apoptosis inducing ligand (TRAIL). We have shown that the breast cancer cell line MDA-MB-231 produces a sufficient amount of osteoprotegerin to bind TRAIL, resulting in an upregulation of receptor activator factor kappa B ligand (RANKL) expression. In conclusion, the presence of osteoprotegerin, as secreted by this cell line, acting as a paracrine factor, could affect breast cancer RANKL production inducing an enhancement of osteolysis and the perpetuation of a vicious cycle. A better understanding of the complex tumor cell-host cell interactions in the bone microenvironment, and of the autocrine and paracrine effects of the secreted (from tumor cells) and released (from bone matrix) factors may facilitate development of effective strategies to inhibit disease progression.

Topics: Bone and Bones; Breast Neoplasms; Cell Line, Tumor; Estrogens; Female; Humans; Osteolysis; Osteoprotegerin; Paracrine Communication; RANK Ligand; TNF-Related Apoptosis-Inducing Ligand; Up-Regulation

The tropism of breast cancer cells for bone and their tendency to induce an osteolytic phenotype are a result of interactions between breast cancer cells and stromal cells and are of paramount importance for bone metastasis. However, the underlying molecular mechanisms remain poorly understood. We hypothesize that tumor-stromal interaction alters gene expression in malignant tumor cells and stromal cells creating a unique expression signature that promotes osteolytic breast cancer bone metastasis and that inhibition of such interactions can be developed as targeted therapeutics. Microarray analysis was performed to investigate gene expression profiling at the tumor-bone (TB) interface versus the tumor alone area from syngenic mice injected with three different syngenic mammary tumor cell lines that differ in their metastatic potential. We identified matrix metalloproteinase 13 (MMP13), receptor activator of NF-kappaB ligand (RANKL), and integrins binding sialoprotein to be genes upregulated at the TB interface and validated. To determine the functional role of MMP13 in tumor-induced osteolysis, mice with Cl66 mammary tumors were treated with MMP13 antisense oligonucleotides (MMP13-ASO) or control scrambled oligonucleotides (control-ASO). Knockdown of MMP13 expression at the TB interface leads to significant reduction in bone destruction and in the number of activated osteoclasts at the TB interface. Further analysis to evaluate the mechanism of MMP13-dependent osteolytic bone metastasis revealed that MMP13-ASO treatment decreased active MMP9, RANKL levels, and transforming growth factor-beta signaling at the TB interface. Together, our data indicate that upregulation of MMP13 at the TB interface is important in tumor-induced osteolysis and suggest that MMP13 is a potential therapeutic target for breast cancer bone metastasis.

Topics: Adenocarcinoma; Animals; Bone and Bones; Bone Neoplasms; Cell Line, Tumor; Enzyme Activation; Female; Gene Expression Profiling; Mammary Neoplasms, Experimental; Matrix Metalloproteinase 13; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred BALB C; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Exogenous osteoprotegerin (OPG) gene modification appears a therapeutic strategy for osteolytic aseptic loosening. The feasibility and efficacy of a cell-based OPG gene delivery approach were investigated using a murine model of knee prosthesis failure. A titanium pin was implanted into mouse proximal tibia to mimic a weight-bearing knee arthroplasty, followed by titanium particles challenge to induce periprosthetic osteolysis. Mouse fibroblast-like synoviocytes were transduced in vitro with either AAV-OPG or AAV-LacZ before transfused into the osteolytic prosthetic joint 3 weeks post surgery. Successful transgene expression at the local site was confirmed 4 weeks later after killing. Biomechanical pullout test indicated a significant restoration of implant stability after the cell-based OPG gene therapy. Histology revealed that inflammatory pseudo-membranes existed ubiquitously at bone-implant interface in control groups, whereas only observed sporadically in OPG gene-modified groups. Tartrate-resistant acid phosphatase+osteoclasts and tumor necrosis factor α, interleukin-1β, CD68+ expressing cells were significantly reduced in periprosthetic tissues of OPG gene-modified mice. No transgene dissemination or tumorigenesis was detected in remote organs and tissues. Data suggest that cell-based ex vivo OPG gene therapy was comparable in efficacy with in vivo local gene transfer technique to deliver functional therapeutic OPG activities, effectively halted the debris-induced osteolysis and regained the implant stability in this model.

Topics: Acid Phosphatase; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Genetic Therapy; Genetic Vectors; Isoenzymes; Knee Prosthesis; Mice; Mice, Inbred BALB C; Osteolysis; Osteoprotegerin; Prosthesis Failure; Tartrate-Resistant Acid Phosphatase; Weight-Bearing

cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cells and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-beta. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Interleukin-17; Matrix Metalloproteinases; Mice; Mice, Nude; Neoplasm Metastasis; Osteolysis; Osteoprotegerin; Parathyroid Hormone-Related Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Micro-structural changes associated with ultra high molecular weight polyethylene particle (UHMWPE) induced osteolysis, the most frequent cause of aseptic loosening, have been intensively investigated in the mammalian calvarian model by histomorphometry and micro-computed tomography. However, little is known regarding the serological changes that occur during this process.. Serological parameters for bone metabolism [calcium, phosphate, osteocalcin (OCN), deoxypyridinoline (DPD)/creatinine, alkaline phosphatase, osteoprotegerin and receptor activator of nuclear factor-κB] were analyzed in this animal model for particle induced osteolysis. Ten C57BL/6 mice were divided at random into sham operated and UHM-WPE implanted groups. Blood and urine samples were collected prior to and at 14 days after surgery.. Implantation of UHMWPE lead to a significant decrease in bone volume (p=0.027). Both groups (sham/UHMWPE) showed a significant increase in calcium (p=0.004/p=0.027) and phosphate (p=0.001/p=0.001), without correlation to particle implantation. Significantly higher concentrations of DPD/creatinine (p=0.034) and OCN (p=0.022) were found after implantation of UHM-WPE. In addition, parameters could not be correlated to particle induced osteolysis.. DPD can be regarded as a valuable parameter for detecting UHMWPE induced osteolysis in the calvarian model. Further studies of serum parameters should focus on the clinical relevance in aseptic prosthetic loosening.

Topics: Alkaline Phosphatase; Amino Acids; Animals; Biomarkers; Calcium; Male; Mice; Mice, Inbred C57BL; Osteocalcin; Osteolysis; Osteoprotegerin; Phosphates; Polyethylene; Postoperative Period; Preoperative Period; RANK Ligand; Time Factors; X-Ray Microtomography

Breast cancer frequently metastasizes to bone, where tumor cells induce osteoclasts to locally destroy bone. During bone resorption, growth factors are locally released that may support bone metastatic growth. Differently from most other tissues, drugs that can limit local turnover, such as bisphosphonates and osteoprotegerin (OPG), are available for bone. We examined the hypothesis that inhibition of bone resorption by two different mechanisms may also affect the growth of cancer cells in bone. For this, we tested the effects of high doses of OPG and zoledronic acid (ZOL) on progression of MDA-231-B/Luc+ breast cancer cells in the bone microenvironment using whole body bioluminescent reporter imaging (BLI). Both treatments significantly inhibited the development of radiographically detectable osteolytic lesions. Histologic examination corroborated the radiographic findings, showing that both treatments preserved the integrity of bone trabeculae and prevented bone destruction (significantly higher trabecular bone volumes vs. vehicle). However, whereas practically no TRAcP-positive osteoclasts were observed in tibiae preparations of animals treated with Fc-OPG, TRAcP-positive osteoclasts were still present in the animals treated with ZOL. Intra-bone tumor burden was reduced with ZOL and Fc-OPG treatment. Although there appeared to be a trend for less overall total tumor burden upon treatment with both compounds, this was not significant as assessed by BLI and histomorphometric analysis due to the extramedullary growth of cancer cells which was not affected by these treatments. Collectively, anti-resorptive agents with different mechanisms of action - ZOL and OPG - significantly reduced cancer-induced osteolysis and intra-osseous tumor burden, but failed to restrain local tumor growth. However, interference with the bone micro-environmental growth support could still be of therapeutic relevance when given to patients early in the course of bone metastatic disease.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Resorption; Breast Neoplasms; Cell Count; Cell Line, Tumor; Cell Proliferation; Diphosphonates; Female; Humans; Imidazoles; Isoenzymes; Luminescent Measurements; Mice; Mice, Inbred BALB C; Mice, Nude; Osteoclasts; Osteolysis; Osteoprotegerin; Radiography; Tartrate-Resistant Acid Phosphatase; Whole Body Imaging; Zoledronic Acid

Topics: Animals; Bone Morphogenetic Protein 4; Bone Remodeling; Calcinosis; Humans; Muscle, Smooth, Vascular; Osteolysis; Osteoprotegerin; RANK Ligand; Rats; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction; Vascular Diseases

It was recently shown that physiological bone remodeling depends on the dynamic balance of two cytokines that are predominantly secreted by osteoblasts. RANKL promotes the differentiation of osteoclastic precursors and the activation of osteoclasts, whereas osteoprotegerin (OPG)inhibits RANKL action. During the development of many tumors, enhanced osteolysis results in pathological bone destruction. Tumor-associated osteolysis is characterized by the degradation and inhibition of osteoprotegerin (OPG) and increased RANKL expression and secretion by tumor tissue. The resulting RANKL/OPG imbalance causes increased generation and activation of osteoclasts and, finally, a significant decrease in bone mass and pathological bone fractures. Tumor cells may also produce many other factors which affect the RANK/RANKL/OPG system and accelerate osteolysis, including IL-1, IL-6, TNF, and MIP-1alpha. The elucidation of the key mechanisms of tumor osteolysis has led to clinical trials of biological therapies based on the inhibition of RANKL and stimulation of OPG activity.

Topics: Humans; Neoplasms; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B

Using an in vivo adeno-associated virus (AAV)-mediated gene transfer technique, this study evaluated the therapeutic effects of an osteoprotegerin (OPG) transgene against orthopaedic wear debris-induced osteolysis in a long-term murine model. A titanium pin was surgically implanted into proximal tibia of Balb/c mice to mimic a weight-bearing knee arthroplasty, followed by an intra-articular challenge with Ti particles to provoke periprosthetic inflammation and osteolysis. rAAV-hOPG or AAV-LacZ vectors were injected into the prosthetic joint at 3 weeks post-op. The tissues were harvested at 2, 4, 12 and 24 weeks after transduction for histological and molecular analyses. Successful transgene expression at the local site was confirmed by real-time PCR and ELISA. Inflammatory pseudo-membranes were ubiquitously present at the interface between the Ti implant and the surrounding bone in both LacZ and virus-free control groups, while soft tissue was only observed sporadically at the bone-implant interface in the OPG group. A significant reduction in TRAP+ osteoclast numbers was observed in the OPG treatment group. MicroCT assessment indicated a marked reversal in the loss of peri-implant bone mineral density (BMD) in the OPG-transduced group, when compared with the LacZ and virus-free controls. Further, OPG gene modification appeared to reduce local bone collagen loss by a mean of 40%. Real-time PCR examination confirmed that in vivo OPG gene transfer dramatically influenced the periprosthetic tissue gene expression profiles by diminishing the mRNA expression of TNF, IL-1, CPK and RANKL. There were no transgene-associated toxic effects apparent during the experiment, and the PCR detection of transgenes in remote organs such as lungs, kidneys, liver, and muscle of contralateral limb were consistently negative. Overall, rAAV-mediated OPG gene transfer effectively reversed Ti-particle-induced bone resorption in this experimental model. The therapeutic effects may be due to the blockage of local osteoclastogenesis and possibly the down-regulation of RANKL expression.

Topics: Animals; Dependovirus; Interleukin-1; Knee Prosthesis; Mice; Mice, Inbred BALB C; Osteolysis; Osteoprotegerin; Prosthesis Design; Prosthesis Failure; RANK Ligand; RNA, Messenger; Time Factors; Tissue Distribution; Titanium; Transgenes; Tumor Necrosis Factor-alpha

Most breast cancer metastases in bone form osteolytic lesions, but the mechanisms of tumor-induced bone resorption and destruction are not fully understood. Although it is well recognized that Wnt/beta-catenin signaling is important for breast cancer tumorigenesis, the role of this pathway in breast cancer bone metastasis is unclear. Dickkopf1 (Dkk1) is a secreted Wnt/beta-catenin antagonist. In the present study, we demonstrated that activation of Wnt/beta-catenin signaling enhanced Dkk1 expression in breast cancer cells and that Dkk1 overexpression is a frequent event in breast cancer. We also found that human breast cancer cell lines that preferentially form osteolytic bone metastases exhibited increased levels of Wnt/beta-catenin signaling and Dkk1 expression. Moreover, we showed that breast cancer cell-produced Dkk1 blocked Wnt3A-induced osteoblastic differentiation and osteoprotegerin (OPG) expression of osteoblast precursor C2C12 cells and that these effects could be neutralized by a specific anti-Dkk1 antibody. In addition, we found that breast cancer cell conditioned media were able to block Wnt3A-induced NF-kappaB ligand reduction in C2C12 cells. Finally, we demonstrated that conditioned media from breast cancer cells in which Dkk1 expression had been silenced via RNAi were unable to block Wnt3A-induced C2C12 osteoblastic differentiation and OPG expression. Taken together, these results suggest that breast cancer-produced Dkk1 may be an important mechanistic link between primary breast tumors and secondary osteolytic bone metastases.

Topics: beta Catenin; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; NF-kappa B; Osteoblasts; Osteolysis; Osteoprotegerin; RANK Ligand; Up-Regulation; Wnt Proteins; Wnt3 Protein; Wnt3A Protein

Tumor cells induce excessive osteoclastogenesis, mediating pathologic bone resorption and subsequent release of growth factors and calcium from bone matrix, resulting in a "vicious cycle" of bone breakdown and tumor proliferation. RANK ligand (RANKL) is an essential mediator of osteoclast formation, function, and survival. In metastatic prostate cancer models, RANKL inhibition directly prevents osteolysis via blockade of osteoclastogenesis and indirectly reduces progression of skeletal tumor burden by reducing local growth factor and calcium concentrations. Docetaxel, a well-established chemotherapy for metastatic hormone-refractory prostate cancer, arrests the cell cycle and induces apoptosis of tumor cells. Suppression of osteoclastogenesis through RANKL inhibition may enhance the effects of docetaxel on skeletal tumors. We evaluated the combination of the RANKL inhibitor osteoprotegerin-Fc (OPG-Fc) with docetaxel in a murine model of prostate cancer bone metastasis. Tumor progression, tumor area, and tumor proliferation and apoptosis were assessed. OPG-Fc alone reduced bone resorption (P < 0.001 versus PBS), inhibited progression of established osteolytic lesions, and reduced tumor area (P < 0.0001 versus PBS). Docetaxel alone reduced tumor burden (P < 0.0001 versus PBS) and delayed the development of osteolytic lesions. OPG-Fc in combination with docetaxel suppressed skeletal tumor burden (P = 0.0005) and increased median survival time by 16.7% (P = 0.0385) compared with docetaxel alone. RANKL inhibition may enhance docetaxel effects by increasing tumor cell apoptosis as evident by increased active caspase-3. These studies show that inhibition of RANKL provides an additive benefit to docetaxel treatment in a murine model of prostate cancer bone metastasis and supports clinical evaluation of this treatment option in patients.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Docetaxel; Humans; Male; Mice; Mice, Nude; Osteolysis; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand; Receptors, Fc; Survival Analysis; Taxoids; Tumor Burden; Whole Body Imaging; Xenograft Model Antitumor Assays

To investigate the expression of bone resorption regulators (receptor activator of nuclear factor kappa B [RANK], RANK ligand [RANKL], and osteoprotegerin [OPG]) in calcifying cystic odontogenic tumor (CCOT), adenomatoid odontogenic tumor (AOT), calcifying epithelial odontogenic tumor (CEOT), odontogenic myxoma (OM), and ameloblastic fibroma (AF).. The expression of these mediators was evaluated by means of immunohistochemistry.. All specimens demonstrated positive immunoreactivity to RANK, RANKL, and OPG. The quantification of these mediators in epithelium revealed a similar pattern of expression for RANKL and OPG in CCOT, AOT, CEOT, and AF. With regard to stromal/mesenchymal cells, the majority of AOT and CCOT cases showed a higher content of OPG than RANKL, whereas CEOT, OM, and especially AF had a tendency to present a greater content of RANKL than OPG.. Our data indicate that the CCOT, AOT, CEOT, OM, and AF cell constituents express key regulators of bone metabolism that might locally modulate tumor-associated bone resorption.

Topics: Adult; Alveolar Bone Loss; Female; Humans; Immunohistochemistry; Jaw Neoplasms; Male; Odontogenic Tumors; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Young Adult

Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) plays an important role in host defense, as well as in inflammation-induced tissue lesions. Here we evaluated the role of NO in bone loss in bacterial infection-induced apical periodontitis by using iNOS-deficient mice (iNOS(-/-)). The iNOS(-/-) mice developed greater inflammatory cell recruitment and osteolytic lesions than WT mice. Moreover, tartrate-resistant acid-phosphatase-positive (TRAP(+)) osteoclasts were significantly more numerous in iNOS(-/-) mice. Furthermore, the increased bone resorption in iNOS(-/-) mice also correlated with the increased expression of receptor activator NF-kappaB (RANK), stromal-cell-derived factor-1 alpha (SDF-1 alpha/CXCL12), and reduced expression of osteoprotegerin (OPG). These results show that NO deficiency was associated with an imbalance of bone-resorption-modulating factors, leading to severe infection-stimulated bone loss.

Topics: Acid Phosphatase; Actinomycosis; Alveolar Bone Loss; Animals; Bacterial Infections; Bacteroidaceae Infections; Biomarkers; Cell Count; Cell Movement; Chemokine CXCL12; Dental Pulp Exposure; Isoenzymes; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Nitric Oxide; Nitric Oxide Synthase Type II; Osteoclasts; Osteolysis; Osteoprotegerin; Periapical Periodontitis; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Tartrate-Resistant Acid Phosphatase

Osteolytic bone disease (OBD) in multiple myeloma (MM) is caused by interactions between MM cells and the bone marrow microenvironment and is characterized by increased osteoclastic bone resorption and decreased osteoblastic bone formation. Recently, the role of osteoblast inhibition has come into focus, especially the possible role of overexpression of DKK1, an inhibitor of the Wnt signalling pathway. Further, CKS2, PSME2 and DHFR have also been reported as candidate genes for OBD. We studied the gene expression by quantitative reverse transcription polymerase chain reaction of TNFSF11 (RANKL), TNFSF11A (RANK), TNFRSF11B (OPG), CCL3 (MIP1A), CCL4 (MIP1B), PTHR1 (PTHrp), DKK1, CKS2, PSME2 and DHFR in purified, immunophenotypic FACS-sorted plasma cells from 171 newly diagnosed MM patients, 20 patients with monoclonal gammopathy of undetermined significance and 12 controls. The gene expressions of the analysed genes were correlated with radiographically assessed OBD. Only overexpression of DKK1 was correlated to the degree of OBD. Myeloma cells did not express TNFSF11A, TNFSF11, or TNFRSF11B, and very rarely expressed CCL3 and PTHR11. CCL4, CKS2, PSME2 and DHFR were variably expressed, but the expression of these genes showed no correlation with OBD. In contrast, loss of PSME2 expression in MM plasma cells was significantly correlated with OBD.

Topics: Adult; Aged; Aged, 80 and over; Carrier Proteins; CDC2-CDC28 Kinases; Cell Cycle Proteins; Chemokine CCL3; Chemokine CCL4; Female; Gene Expression; Humans; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Multiple Myeloma; Muscle Proteins; Osteolysis; Osteoprotegerin; Plasma Cells; Proteasome Endopeptidase Complex; Protein Kinases; Receptor Activator of Nuclear Factor-kappa B; Receptor, Parathyroid Hormone, Type 1; Tetrahydrofolate Dehydrogenase

Metastases to bone are a frequent complication of human prostate cancer and result in the development of osteoblastic lesions that include an underlying osteoclastic component. Previous studies in rodent models of breast and prostate cancer have established that receptor activator of NF-kappaB ligand (RANKL) inhibition decreases bone lesion development and tumor growth in bone. RANK is essential for osteoclast differentiation, activation, and survival via its expression on osteoclasts and their precursors. RANK expression has also been observed in some tumor cell types such as breast and colon, suggesting that RANKL may play a direct role on tumor cells.. Male CB17 severe combined immunodeficient (SCID) mice were injected with PC3 cells intratibially and treated with either PBS or human osteprotegerin (OPG)-Fc, a RANKL antagonist. The formation of osteolytic lesions was analyzed by X-ray, and local and systemic levels of RANKL and OPG were analyzed. RANK mRNA and protein expression were assessed on multiple prostate cancer cell lines, and events downstream of RANK activation were studied in PC3 cells in vitro.. OPG-Fc treatment of PC3 tumor-bearing mice decreased lesion formation and tumor burden. Systemic and local levels of RANKL expression were increased in PC3 tumor bearing mice. PC3 cells responded to RANKL by activating multiple signaling pathways which resulted in significant changes in expression of genes involved in osteolysis and migration. RANK activation via RANKL resulted in increased invasion of PC3 cells through a collagen matrix.. These data demonstrate that host stromal RANKL is induced systemically and locally as a result of PC3 prostate tumor growth within the skeleton. RANK is expressed on prostate cancer cells and promotes invasion in a RANKL-dependent manner.

Topics: Animals; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, SCID; Neoplasm Transplantation; Osteolysis; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; RNA, Messenger; Signal Transduction

The aim of this study was to compare the expression of nuclear factor kappaB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) in odontogenic epithelial tumors and dentigerous cysts.. The expression of these molecules was evaluated in solid/multicystic ameloblastomas (n = 12) and unicystic ameloblastomas (n = 8), keratocystic odontogenic tumors (n = 19), dentigerous cysts (n = 9), and dental follicles (n = 9) by immunohistochemistry.. In odontogenic epithelium, a similar expression of RANK, RANKL, and OPG was observed in all samples. With regard to stroma, the number of RANK-positive and RANKL-positive cells was higher in solid/multicystic ameloblastomas compared with dentigerous cysts. Dental follicles had lower numbers of RANK-positive, RANKL-positive, and OPG-positive cells than solid/multicystic ameloblastomas and keratocystic odontogenic tumors. The majority of solid/multicystic ameloblastomas (75%) and unicystic ameloblastomas (62.5%) had higher numbers of RANKL-positive than OPG-positive cells. In contrast, 62.4% of keratocystic odontogenic tumors and 100% of dentigerous cysts exhibited a higher content of OPG-positive than RANKL-positive cells.. Our results indicate differences in the RANK, RANKL, and OPG expression in odontogenic epithelial tumors. The imbalance of these factors could contribute to the differential bone/tooth resorption activity in these lesions.

Topics: Ameloblastoma; Cell Count; Dentigerous Cyst; Epithelial Cells; Gene Expression; Humans; Jaw Neoplasms; NF-kappa B; Odontogenic Tumors; Osteolysis; Osteoprotegerin; RANK Ligand; Root Resorption; Statistics, Nonparametric; Stromal Cells

Multiple myeloma (MM) is characterized by osteolytic bone lesions (OBL) that arise as a consequence of osteoblast inactivation and osteoclast activation adjacent to tumor foci within bone. Wnt signaling in osteoblasts regulates osteoclastogenesis through the differential activation and inactivation of Receptor Activator of Nuclear factor Kappa B Ligand (RANKL) and osteoprotegerin (OPG), positive and negative regulators of osteoclast differentiation, respectively. We demonstrate here that MM cell-derived DKK1, a soluble inhibitor of canonical Wnt signaling, disrupted Wnt3a-regulated OPG and RANKL expression in osteoblasts. Confirmed in multiple independent assays, we show that pretreatment with rDKK1 completely abolished Wnt3a-induced OPG mRNA and protein production by mouse and human osteoblasts. In addition, we show that Wnt3a-induced OPG expression was diminished in osteoblasts cocultured with a DKK1-expressing MM cell line or primary MM cells. Finally, we show that bone marrow sera from 21 MM patients significantly suppressed Wnt3a-induced OPG expression and enhanced RANKL expression in osteoblasts in a DKK1-dependent manner. These results suggest that DKK1 may play a key role in the development of MM-associated OBL by directly interrupting Wnt-regulated differentiation of osteoblasts and indirectly increasing osteoclastogenesis via a DKK1-mediated increase in RANKL-to-OPG ratios.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Line; Cell Line, Tumor; Coculture Techniques; DNA Primers; Gene Expression; Gene Silencing; Humans; Intercellular Signaling Peptides and Proteins; Mice; Multiple Myeloma; Osteoblasts; Osteolysis; Osteoprotegerin; RANK Ligand; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Transfection; Tumor Cells, Cultured; Wnt Proteins; Wnt3 Protein; Wnt3A Protein

Aseptic loosening of total joint replacements is caused by wear debris-induced osteoclastic bone resorption, for which bisphosphonates (BPs) and RANK antagonists have been developed. Although BPs are effective in preventing metabolic bone loss, they are less effective for inflammatory bone loss. Because this difference has been attributed to the antiapoptotic inflammatory signals that protect osteoclasts from BP-induced apoptosis, but not RANK antagonists, we tested the hypothesis that osteoprotegerin (OPG) is more effective in preventing wear debris-induced osteolysis than zoledronic acid (ZA) or alendronate (Aln) in the murine calvaria model using in vivo micro-CT and traditional histology. Although micro-CT proved to be incompatible with titanium (Ti) particles, we were able to demonstrate a 3.2-fold increase in osteolytic volume over 10 days induced by polyethylene (PE) particles versus sham controls (0.49 +/- 0.23 mm(3) versus 0.15 +/- 0.067 mm(3); p < 0.01). Although OPG and high-dose ZA completely inhibited this PE-induced osteolysis (p < 0.001), pharmacological doses of ZA and Aln were less effective but still reached statistical significance (p < 0.05). Traditional histomorphometry of the sagital suture area of calvaria from both Ti and PE-treated mice confirmed the remarkable suppression of resorption by OPG (p < 0.001) versus the lack of effect by physiological BPs. The differences in drug effects on osteolysis were largely explained by the significant difference in osteoclast numbers observed between OPG versus BPs in both Ti- and PE-treated calvaria; and linear regression analyses that demonstrated a highly significant correlation between osteolysis volume and sagittal suture area versus osteoclast numbers (p < 0.001).

Topics: Alendronate; Animals; Biocompatible Materials; Bone Density Conservation Agents; Diphosphonates; Disease Models, Animal; Image Processing, Computer-Assisted; Imidazoles; Mice; Osteoclasts; Osteolysis; Osteoprotegerin; Polyethylene; Prosthesis Failure; Skull; Surface Properties; Tomography, X-Ray Computed; Zoledronic Acid

Protein expression of osteopontin (OPN), osteoprotegerin (OPG), bone sialoprotein (BSP), osteocalcin (OC), RANKL and PTHrP was determined by use of immunohistochemical analysis on tissue arrays (48 cases of PVNS, 20 cases of active (a-RA), non-active rheumatoid arthritis (na-RA), and osteoarthritis (OA)). Additionally, gene expression was analysed using complimentary DNA (cDNA) microarrays. All PVNS cases showed a higher level of both protein and gene expression of RANKL, OPN and BSP in comparison with OA cases. Expression of OPG was not significantly different in PVNS compared to OA. The RANKL/OPG expression ratio was significantly higher in PVNS than in OA. High expressions level of proteins involved in bone degradation in PVNS may promote an intra-osseous propagation of the lesion. This evidence suggests that PVNS might respond to treatment using specific inhibitors of RANKL, OPN and BSP.

Topics: Adult; Aged; Biomarkers; Female; Humans; Immunohistochemistry; Integrin-Binding Sialoprotein; Male; Middle Aged; Osteoarthritis; Osteolysis; Osteopontin; Osteoprotegerin; RANK Ligand; RNA; Sialoglycoproteins; Synovitis, Pigmented Villonodular; Tissue Array Analysis

The skeletal system is the most frequent metastatic site of hematogenous spread of urologic carcinomas. Osseus metastases are classified as osteoneutral, osteolytic, osteoblastic and combinations thereof. Osteolytic metastases lead to bone resorption by activating osteoclasts, while osteoblastic metastases stimulate osteoblasts by paracrine mechanisms. The local osteoblastic effect is associated with secondary systemic bone resorption. The use of bisphosphonates is now an established supportive therapy and newer treatment strategies including targeted intervention in the pathophysiology of bone metastases and radioimmunotherapy are being applied or will be coming soon.

Topics: Bone and Bones; Bone Marrow Neoplasms; Bone Neoplasms; Bone Resorption; Cytokines; Diphosphonates; Humans; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; Parathyroid Hormone-Related Protein; Radioimmunotherapy; RANK Ligand; Urologic Neoplasms

Epidermal growth factor (EGF)-like ligands and their receptors constitute one of the most important signaling networks functioning in normal tissue development and cancer biology. Recent in vivo mouse models suggest this signaling network plays an essential role in bone metabolism. Using a coculture system containing bone marrow macrophage and osteoblastic cells, here we report that EGF-like ligands stimulate osteoclastogenesis by acting on osteoblastic cells. This stimulation is not a direct effect because osteoclasts do not express functional EGF receptors (EGFRs). Further studies reveal that EGF-like ligands strongly regulate the expression of two secreted osteoclast regulatory factors in osteoblasts by decreasing osteoprotegerin (OPG) expression and increasing monocyte chemoattractant protein 1 (MCP1) expression in an EGFR-dependent manner and consequently stimulate TRAP-positive osteoclast formation. Addition of exogenous OPG completely inhibited osteoclast formation stimulated by EGF-like ligands, while addition of a neutralizing antibody against MCP-1 exhibited partial inhibition. Coculture with bone metastatic breast cancer MDA-MB-231 cells had similar effects on the expression of OPG and MCP1 in the osteoblastic cells, and those effects could be partially abolished by the EGFR inhibitor PD153035. Because a high percentage of human carcinomas express EGF-like ligands, our findings suggest a novel mechanism for osteolytic lesions caused by cancer cells metastasizing to bone.

Topics: Animals; Bone Neoplasms; Cells, Cultured; Chemokine CCL2; Coculture Techniques; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Humans; Ligands; Mice; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Signal Transduction

Osteosarcoma is the most frequent primary bone tumor that develops mainly in the young, the median age of diagnosis being 18 years. Despite improvement in osteosarcoma treatment, survival rate is only 30% at 5 years for patients with pulmonary metastases at diagnosis. This warrants exploration of new therapeutic options, and among them, osteoprotegerin (OPG), a naturally occurring protein that inhibits bone resorption, is very promising in blocking the vicious cycle between bone resorption and tumor proliferation that takes place during tumor development in bone site. As OPG binds and inhibits the activity of tumor necrosis factor-related apoptosis-inducing ligand, the truncated form of murine OPG 1-194 was used. The cDNA encoding OPG was administered by gene transfer using replication-defective adenoviral vector or was associated with an amphiphilic polymer in two models of rodent osteosarcoma. In both models, OPG gene transfer was effective in preventing the formation of osteolytic lesions associated with osteosarcoma development, in reducing the tumor incidence and the local tumor growth, leading to a 4-fold augmentation of mice survival 28 days postimplantation. On the contrary, OPG did not prevent the development of pulmonary metastasis alone, suggesting that bone environment is necessary for OPG therapeutic efficacy. Because OPG has no direct activity on osteosarcoma cells in vitro (cell binding, cell proliferation, apoptosis, or cell cycle distribution), we show that OPG exerts indirect inhibitory effect on tumor progression through the inhibition of RANKL whose production is enhanced in bone tumor environment, leading to osteolysis inhibition as reflected by osteoclast number decrease.

Topics: Adenoviridae; Animals; Apoptosis; Bone Density; Bone Neoplasms; Bone Resorption; Caspases; Cell Cycle; Cell Proliferation; Enzyme-Linked Immunosorbent Assay; Genetic Therapy; Genetic Vectors; Male; Mice; Mice, Inbred C3H; Myoblasts; Osteolysis; Osteoprotegerin; Osteosarcoma; RANK Ligand; Rats; Transduction, Genetic; Xenograft Model Antitumor Assays

Earlier reports showed that the balance between receptor activator of nuclear factor-kappaB ligand (RANKL) and its decoy-receptor osteoprotegerin (OPG) plays an important role in the pathogenesis of metastatic osteolysis induced by neuroblastoma cells. In this study, we investigated whether circulating levels of OPG, RANKL and their ratio were associated to the presence of osteolytic lesions in advanced neuroblastoma, as well as whether they provided additional information on the severity and prognosis of the disease. Plasma levels of RANKL and OPG were measured in 54 newly diagnosed neuroblastomas; 27 of them showed metastatic disease (stage IV), including 19 bone dissemination. Thirty-five children who were admitted to the pediatric department for minor surgical problems served as control group. OPG was significantly lower in all patients compared with controls, while RANKL levels were significantly increased in advanced neuroblastoma. OPG-to-RANKL ratio decreased in stage-IV patients, and particularly in those who had bone metastases. The diagnostic accuracy of the OPG-to-RANKL ratio in discriminating the presence of osteolytic lesions was not confirmed statistically. OPG correlated significantly with other prognostic factors, namely, ferritin and neurone-specific enolase. In addition, an inverse relationship was found between OPG and event-free survival, and it was more significant in patients who had bone metastasis. This pilot study confirms that the production of OPG and RANKL is disregulated in neuroblastoma. Although the OPG-to-RANKL ratio does not have a predictive value in detecting bone metastasis, the measurement of the previously mentioned markers could be useful in decisions regarding the use of adjuvant therapies.

Topics: Biomarkers, Tumor; Bone and Bones; Bone Neoplasms; Carrier Proteins; Child; Child, Preschool; Disease-Free Survival; Female; Glycoproteins; Humans; Infant; Male; Membrane Glycoproteins; Neoplasm Staging; Neuroblastoma; Osteolysis; Osteoprotegerin; Predictive Value of Tests; Prognosis; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Sensitivity and Specificity

The cellular and humoral mechanisms accounting for osteolysis in skeletal metastases of malignant melanoma are uncertain. Osteoclasts, the specialised multinucleated cells that carry out bone resorption, are derived from monocyte/macrophage precursors. We isolated tumour-associated macrophages (TAMs) from metastatic (lymph node/skin) melanomas and cultured them in the presence and absence of osteoclastogenic cytokines and growth factors. The effect of tumour-derived fibroblasts and melanoma cells on osteoclast formation and resorption was also analysed. Melanoma TAMs (CD14+/CD51-) differentiated into osteoclasts (CD14-/CD51+) in the presence of receptor activator for nuclear factor kappaB ligand (RANKL) and macrophage-colony stimulating factor. Tumour-associated macrophage-osteoclast differentiation also occurred via a RANKL-independent pathway when TAMs were cultured with tumour necrosis factor-alpha and interleukin (IL)-1alpha. RT-PCR showed that fibroblasts isolated from metastatic melanomas expressed RANKL messenger RNA and the conditioned medium of cultured melanoma fibroblasts was found to be capable of inducing osteoclast formation in the absence of RANKL; this effect was inhibited by the addition of osteoprotegerin (OPG). We also found that cultured human SK-Mel-29 melanoma cells produce a soluble factor that induces osteoclast differentiation; this effect was not inhibited by OPG. Our findings indicate that TAMs in metastatic melanomas can differentiate into osteoclasts and that melanoma fibroblasts and melanoma tumour cells can induce osteoclast formation by RANKL-dependent and RANKL-independent mechanisms, respectively.

Topics: Aged; Aged, 80 and over; Antineoplastic Agents; Bone Resorption; Carrier Proteins; Cell Differentiation; Cells, Cultured; Culture Media, Conditioned; Female; Fibroblasts; Glycoproteins; Humans; Interleukin-1; Lymphatic Metastasis; Macrophages; Male; Melanoma; Membrane Glycoproteins; Middle Aged; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms; Tumor Necrosis Factor-alpha

This study investigates receptor activator NF-kappaB (RANK), RANK ligand (RANKL) and tumour necrosis factor (TNFalpha), key factors regulating bone turnover, present in the tissues near peri-prosthetic osteolysis. Tissue was obtained from zones of peri-prosthetic osteolysis from 11 patients undergoing revision of total hip prostheses, analysed preoperatively by high-resolution spiral multislice CT using a metal artefact suppression protocol. Synovial tissue from 10 patients with osteoarthritis undergoing primary hip replacement was used as control tissue. Immunohistochemical analysis of formalin fixed tissue sections demonstrated that RANK, RANKL and TNFalpha were strongly expressed by large multinucleated cells containing polyethylene wear debris in revision tissues. Control tissue stained weakly for RANK, RANKL and TNFalpha. A strong statistical correlation (p<0.02) was found between the five parameters, volume of bone loss, polyethylene wear debris, RANK, RANKL and TNFalpha expression. Importantly, in vitro studies revealed that RANKL and TNFalpha synergise to increase the volume of bone resorbed, by more than seven fold, when compared to the effect of either cytokine treatment alone. This suggests that the interaction of TNFalpha and RANKL promotes osteoclast activity associated with polyethylene wear and therapies targeting TNF activity may be useful to treat peri-implant osteolysis.

Topics: Arthroplasty, Replacement, Hip; Bone Resorption; Carrier Proteins; Glycoproteins; Hip; Hip Prosthesis; Humans; Immunohistochemistry; Membrane Glycoproteins; Osteoclasts; Osteolysis; Osteoprotegerin; Polyethylenes; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tomography, X-Ray Computed; Tumor Necrosis Factor-alpha

Previous studies have suggested that the balance between receptor activator of nuclear factor-kappaB ligand (RANKL) and its decoy-receptor osteoprotegerin (OPG) in local tissue seems to play a crucial role in the loosening of the total hip replacement. The aim of this study was to evaluate whether the circulating levels of OPG and RANKL, as well as their ratio, could be different in patients with aseptic loosening compared with patients with stable implants.. One hundred and twenty-eight subjects were recruited. They included thirty-nine patients with osteoarthritis who had not yet undergone total hip arthroplasty, thirty-three patients who had undergone total hip arthroplasty and had clinically and radiographically stable implants, thirty-six patients with aseptic loosening of total hip arthroplasty components, and twenty healthy volunteers. Serum levels of OPG and RANKL were measured with use of an immunoenzymatic method, and in each individual the OPG-to-RANKL ratio was calculated.. In every group, a significant correlation was detected between OPG concentration and age (r = 0.58, p < 0.0001), especially in individuals older than fifty years, while gender and underlying disease were not found to influence serum levels of the tested parameters. In comparison with the levels in healthy donors and patients with a stable total hip replacement, the serum levels of OPG were increased in the patients who had not yet had an arthroplasty, those with aseptic loosening of a total hip replacement, and those with a cemented total hip replacement. Moreover, the OPG serum level provided good diagnostic accuracy in detecting the implant failure. A correlation was found between the sum of the osteolytic areas seen radiographically around the femoral stem and the RANKL level (r = 0.38, p = 0.02) and the OPG-to-RANKL ratio (r = -0.29, p = 0.04).. An increase in OPG levels may reflect a protective mechanism of the skeleton to compensate for the osteolytic activity that occurs in severe osteoarthritis and in aseptic loosening. Prospective studies are needed to determine whether serum OPG levels could be used as markers for monitoring the stability of the implant, as well as for predicting aseptic loosening.. Diagnostic study, Level III. See Instructions to Authors for a complete description of levels of evidence.

Topics: Adult; Aged; Aged, 80 and over; Arthroplasty, Replacement, Hip; Biomarkers; Carrier Proteins; Case-Control Studies; Female; Glycoproteins; Hip Prosthesis; Humans; Male; Membrane Glycoproteins; Middle Aged; Osteoarthritis, Hip; Osteolysis; Osteoprotegerin; Prosthesis Failure; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

We report a newly recognized bone disorder consisting of polyostotic expansile osteolysis affecting long bones and iliac bones; hyperostosis of the skull, thoracic cage, and medial portion of both clavicles; pectus carinatum; gigantiform synovial masses of the elbows and knees; atrial septal defect; cardiomegaly; unilateral cryptorchidism; and mental deficiency. Affected bones can be grouped into four general types of skeletal pathology: (1) expansile osteolysis, (2) osteolysis without expansion, (3) expansion without osteolysis, and (4) hyperostosis. Some bones remained unaffected. We have named the condition "polyosteolysis/hyperostosis syndrome." It is clearly at variance with any previously reported bone disorder, including familial expansile osteolysis, juvenile Paget disease, and McCune-Albright syndrome (and polyostotic fibrous dysplasia). Because our patient shared some features in common with juvenile Paget disease, we thought that mutational analysis of TNFRSF11B was indicated, even though our patient had some manifestations not found in juvenile Paget disease. Direct sequencing failed to identify a TNFRSF11B mutation. Because the parents of our propositus were first cousins suggests that polyosteolysis/hyperostosis syndrome may possibly have autosomal recessive inheritance.

Topics: Child; Humans; Hyperostosis; Male; Osteolysis; Osteoprotegerin; Syndrome

This study was undertaken to investigate how fibroblasts respond to stimulation with particulate wear debris and/or conditioned media obtained from pathologic tissue, and whether these activated fibroblasts express compounds that are involved in bone resorption.. Conditioned media from explant cultures of synovial tissue, periprosthetic soft tissue (interface membranes), titanium particles, and proinflammatory cytokines were used to stimulate fibroblasts. RNase protection assay was used to measure altered gene expression, and enzyme-linked immunosorbent assay, Western blot hybridization, and flow cytometry were used to determine fibroblast protein expression. Tartrate-resistant acid phosphatase staining was used to identify multinucleated osteoclast-like cells.. The most dominant compounds measured in the conditioned media from interface membranes were tumor necrosis factor alpha (TNFalpha), monocyte chemoattractant protein 1 (MCP-1), interleukin-1beta (IL-1beta), IL-6, IL-8, and vascular endothelial growth factor. Fibroblasts phagocytosed particulate wear debris and responded to cytokine/chemokine stimulation. The most prominent up-regulated genes and proteins secreted by fibroblasts in response to stimulation were matrix metalloproteinase 1, MCP-1, IL-1beta, IL-6, IL-8, cyclooxygenase 1 (COX-1), COX-2, leukemia inhibitory factor 1, transforming growth factor beta1 (TGFbeta1), and TGFbeta receptor type I. In addition, interface membrane fibroblasts expressed RANKL and osteoprotegerin in response to stimulation with conditioned media, TNFalpha, or IL-1beta. Stimulated fibroblasts cocultured with bone marrow cells in the presence of macrophage colony-stimulating factor induced osteoclastogenesis.. Interface membrane fibroblasts respond directly to particulate wear debris, possibly via phagocytosis, expressing proinflammatory cytokines and RANKL. Thus, these cells may be actively involved in osteoclastogenesis and pathologic (periprosthetic) bone resorption.

Topics: Adult; Aged; Aged, 80 and over; Arthroplasty, Replacement, Knee; Bone Resorption; Cells, Cultured; Culture Media, Conditioned; Cytokines; Female; Fibroblasts; Gene Expression Regulation; Humans; Knee Prosthesis; Macrophage Colony-Stimulating Factor; Male; Middle Aged; Osteoclasts; Osteolysis; Osteoprotegerin; Phagocytosis; RANK Ligand; RNA, Messenger; Titanium; Vascular Endothelial Growth Factor A

Bone resorption is regulated by cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta) and by the balance of a receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG). The aim of this study was to investigate the mechanism of particle-induced osteolysis in murine calvariae by assessing the extent of osteolysis and the expression of inflammatory cytokines, RANKL and OPG after implantation of metal and polyethylene particles. The murine calvariae implanted with Ti6Al4V, CoCr or high-density polyethylene (HDP) particles showed significantly more extensive osteolysis and elevated levels of inflammatory cytokines. The ratio between RANKL and OPG was high in the mice implanted with Ti6Al4V and HDP particles, but not in the mice implanted with CoCr particles. These observations suggested that CoCr particle-induced osteoclastogenesis may be caused directly by inflammatory cytokines rather than by the RANKL-RANK pathway. There might be different mechanisms at work in particle-induced osteolysis between Ti6Al4V, HDP and CoCr.

Topics: Animals; Biocompatible Materials; Carrier Proteins; Chromium Alloys; Foreign-Body Reaction; Gene Expression Regulation; Glycoproteins; Materials Testing; Membrane Glycoproteins; Men; Mice; Mice, Inbred C57BL; Microspheres; Osteolysis; Osteoprotegerin; Particle Size; Polyethylene; Prosthesis-Related Infections; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Titanium

Osteoprotegerin (OPG), a critical regulator of osteoclastogenesis, is expressed by prostate cancer cells, and OPG levels are increased in patients with prostate cancer bone metastases. The objective of this study was to investigate the effects of OPG overexpression on prostate cancer cells and prostate cancer/bone cell interactions in vitro and in vivo. OPG-transfected C4-2 cells expressed 8.0 ng OPG per mL per 10(6) cells, whereas no OPG was detected in the media of C4-2 cells transfected with a control plasmid. OPG overexpressed by C4-2 cells protected these cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis and decreased osteoclast formation. Subcutaneous OPG-C4-2 and pcDNA-C4-2 tumors exhibited similar growth and take-rate characteristics. However, when grown in bone, tumor volume was decreased in OPG-C4-2 versus pcDNA-C4-2 (P=0.0017). OPG expressed by C4-2 cells caused increases in bone mineral density (P=0.0074) and percentage of trabecular bone volume (P=0.007), and decreases in numbers of osteoblasts and osteoclasts when compared with intratibial pcDNA-C4-2 tumors (P=0.003 and P=0.019, respectively). In summary, our data show that increased expression of OPG in C4-2 cells does not directly affect proliferation of prostate cancer cells but indirectly decreases growth of C4-2 tumors in the bone environment. Our data also show that OPG expressed by C4-2 cells inhibits bone lysis associated with C4-2 bone metastasis, which results in net increases in bone volume. We therefore hypothesize that OPG expressed in prostate cancer patient bone metastases may be at least partially responsible for the osteoblastic character of most prostate cancer bone lesions.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Bone Neoplasms; Culture Media, Conditioned; Glycoproteins; Humans; Male; Membrane Glycoproteins; Mice; Mice, SCID; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; Prostatic Neoplasms; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA, Messenger; Survival Rate; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Up-Regulation

The highly osteolytic interface tissue between the bone and loosening total hip prosthesis is characterized by low pH, formation of foreign body giant cells, osteoclasts, and production of receptor activator of nuclear factor-kappaB (RANKL) and cathepsin K. We hypothesized that fibroblasts in the interface tissue may form a source for RANKL production.. Primary interface tissue fibroblasts, fibrous joint capsule fibroblasts, and trabecular bone osteoblasts were stimulated with tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), IL-6, IL-11, or 1alpha,25-(OH)2 vitamin D3. Cellular RANKL and released cathepsin K were detected by Western blotting. RANKL in cell lysates and osteoprotegerin (OPG) in cell culture medium were measured by ELISA. RANKL, OPG, and cathepsin K mRNA were measured with quantitative reverse transcriptase polymerase chain reaction.. Interface tissue fibroblasts were found to produce RANKL. 1alpha,25-(OH)2 vitamin D3 stimulation increased RANKL mRNA expression. TNF-alpha was found to be the most potent OPG inducer in interface tissue fibroblasts. Cathepsin K mRNA production in fibroblasts was upregulated roughly 3-fold (p < 0.01) after 1alpha,25-(OH)2D3 stimulation, and both pro- and active cathepsin K protein was released to fibroblast culture media.. Interface tissue fibroblasts are able to produce RANKL, OPG, and cathepsin K and may contribute indirectly and directly to pathologic periprosthetic collagenolysis and bone destruction.

Topics: Aged; Arthroplasty, Replacement, Hip; Calcitriol; Carrier Proteins; Cathepsin K; Cathepsins; Cells, Cultured; Culture Media, Conditioned; Cytokines; Female; Fibroblasts; Glycoproteins; Hip Prosthesis; Humans; Male; Membrane Glycoproteins; Osteolysis; Osteoprotegerin; Prosthesis Failure; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Both macrophages and fibroblasts are the main cell types found in periprosthetic tissues surrounding failed joint arthroplasties. These fibroblasts are known to express RANKL and to produce TNFalpha, factors which promote osteoclast formation and bone resorption. In this study we have analysed the role that arthroplasty membrane-derived fibroblasts (AFb) play in inducing the generation of bone resorbing osteoclasts.. Fibroblasts were isolated from periprosthetic tissues and co-cultured with human monocytes in an osteoclast differentiation assay in the presence or absence of M-CSF and inhibitors of RANKL (OPG) and/or TNFalpha. RANKL expression by AFbs was determined by RT-PCR and the extent of osteoclast differentiation by the expression of TRAP, VNR and evidence of lacunar resorption.. In the presence of M-CSF, large numbers of TRAP(+) and VNR(+) multinucleated cells capable of lacunar resorption, were noted in co-cultures of monocytes and RANKL-expressing AFbs. Cell-cell contact was required for osteoclast formation. The addition of OPG and anti-TNFalpha alone significantly reduced but did not abolish the extent of osteoclast formation, whereas the addition of both together abolished osteoclast formation and lacunar resorption.. Our results indicate that fibroblasts in periprosthetic tissues are capable of inducing the differentiation of normal human peripheral blood mononuclear cells to mature osteoclasts by a mechanism that involves both RANKL and TNFalpha. Suppression of both RANKL and inflammatory cytokines is likely to be required to control periprosthetic osteolysis.

Topics: Aged; Aged, 80 and over; Arthroplasty; Carrier Proteins; Female; Fibroblasts; Glycoproteins; Humans; Male; Membrane Glycoproteins; Middle Aged; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tumor Necrosis Factor-alpha

We developed a rodent model that mimics the osteoblastic and osteolytic changes associated with human metastatic prostate cancer. Microarray analysis identified MMP-7, cathepsin-K, and apolipoprotein D as being upregulated at the tumor-bone interface. MMP-7, which was produced by osteoclasts at the tumor-bone interface, was capable of processing RANKL to a soluble form that promoted osteoclast activation. MMP-7-deficient mice demonstrated reduced prostate tumor-induced osteolysis and RANKL processing. This study suggests that inhibition of MMP-7 will have therapeutic benefit in the treatment of prostate cancer-induced osteolysis.

Topics: Acid Phosphatase; Actins; Animals; Carrier Proteins; Disease Models, Animal; Down-Regulation; Gene Expression; Gene Expression Profiling; Glycoproteins; Humans; Isoenzymes; Male; Matrix Metalloproteinase 7; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Biological; Monocytes; Osteoclasts; Osteolysis; Osteoprotegerin; Parathyroid Hormone-Related Protein; Prostatic Neoplasms; RANK Ligand; Rats; Rats, Inbred F344; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Skull; Tartrate-Resistant Acid Phosphatase; Up-Regulation

Zoledronic acid (ZA) has been shown to inhibit prostate tumor growth in vitro and have beneficial effects in patients with advanced prostate cancer (CaP). The aim of this study was to determine whether ZA exhibits direct anti-tumor effects on CaP cells in vivo. To distinguish the effects of inhibition of osteolysis and direct anti-tumor activity of ZA in vivo, we compared the results of treatment with ZA and osteoprotegerin (Fc-OPG), which inhibits osteolysis, but without significant direct anti-tumor effects. In vitro Fc-OPG had no significant effects on C4-2 proliferation, whereas ZA decreased proliferation. However, both agents decreased tumor growth in bone. Moreover, both increased bone volume and prevented the overall decreases in BMD associated with growth of C4-2 cells in bone. Our study provides novel and significant observations that the in vivo effects of ZA are consistent with indirect effects mediated by osteoclasts.

Topics: Animals; Antibodies; Antineoplastic Agents; Apoptosis; Bone Density; Bone Density Conservation Agents; Bone Neoplasms; Cell Proliferation; Densitometry; Diphosphonates; Glycoproteins; Humans; Imidazoles; Immunoglobulin Fc Fragments; In Vitro Techniques; Male; Mice; Mice, SCID; Neoplasm Metastasis; Osteoclasts; Osteolysis; Osteoprotegerin; Prostatic Neoplasms; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tibia; Time Factors; Zoledronic Acid

Adenocarcinoma of the prostate exhibits a clear propensity for bone and is associated with the formation of osteoblastic metastases. It has previously been suggested that osteoclast activity may be necessary for the development of these osteoblastic metastases based on data from lytic and mixed lytic-blastic tumors. Here we investigate the effects of complete in vivo osteoclast depletion via the blockade of receptor activator of NF:kappaB (RANK) on the establishment and progression of purely osteoblastic (LAPC-9 cells) bone lesions induced by human prostate cancer cells using a SCID mouse intratibial injection model. The subcutaneous administration of the RANK antagonist (15 mg/kg) RANK:Fc did not prevent the formation of purely osteoblastic lesions, indicating that osteoclasts may not be essential to the initial development of osteoblastic metastases. However, RANK:Fc protein appeared to inhibit the progression of established osteoblastic lesions, suggesting that osteoclasts may be involved in the subsequent growth of these tumors once they are already present. In contrast, RANK:Fc treatment effectively blocked the establishment and progression of purely osteolytic lesions formed by PC-3 cells, which served as a positive control. These results indicate that in vivo RANK blockade may not be effective for the prevention of osteoblastic metastasis but may potentially represent a novel therapy that limits the growth of established metastatic CaP lesions in bone.

Topics: Animals; Bone Neoplasms; Glycoproteins; Immunoglobulin Fc Fragments; Male; Mice; Mice, SCID; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; Prostatic Neoplasms; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Fusion Proteins

Alterations of the key regulators of osteoclastogenesis, receptor activator of NF-kappaB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) have been implicated in wear particle-induced osteolysis, the most common cause for implant failure in total joint replacements. This study investigated the effect of exogenous OPG on ultra-high-molecular-weight polyethylene (UHMWPE) particle-induced osteolysis. The murine calvarial osteolysis model was utilized in 28 C57BL/6J mice randomized to four groups. Group I underwent sham surgery only, group II received UHMWPE particles, and group III and IV particles and subcutaneous OPG starting from day 0 (group III) or day 5 (group IV) until sacrifice. After 2 weeks, calvaria were prepared for histology and histomorphometry. Bone resorption was measured within the midline suture using Giemsa staining and osteoclast numbers were determined using TRAP staining. UHMWPE particle implantation resulted in grossly pronounced osteoclastogenesis and bone resorption. Both immediate and delayed treatment with OPG counteracted these particle-induced effects significantly, suppressing osteoclast formation and bone resorption (p < 0.001 and p < 0.001, respectively). In conclusion, exogenous OPG markedly suppressed UHMWPE particle-induced osteolysis in a murine calvarial model. This important finding underscores the crucial significance of the OPG-RANKL-RANK signaling in wear particle-induced osteolysis. Exogenous OPG may prove an effective treatment modality for wear debris-mediated periprosthetic osteolysis after total joint arthroplasty.

Topics: Animals; Biocompatible Materials; Female; Glycoproteins; Male; Materials Testing; Mice; Mice, Inbred C57BL; Models, Biological; Osteoclasts; Osteolysis; Osteoprotegerin; Particle Size; Polyethylenes; Prosthesis Failure; Random Allocation; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Skull

Giant-cell tumor of bone (GCTB) and giant-cell tumor of soft tissue (GCTST) are tumors that contain a prominent osteoclastlike giant-cell component. The precise relationship between these morphologically similar tumors is unclear, and the cellular mechanism whereby giant cells accumulate within these and other locally aggressive tumors is uncertain. In this study, we have examined the cytochemical, functional, and molecular phenotype of the mononuclear and multinucleated components of GCTB and GCTST. Giant cells in GCTB and GCTST exhibited an osteoclast phenotype expressing tartrate-resistant acid phosphatase and vitronectin receptor and being capable of lacunar resorption. The mononuclear stromal cells derived from GCTB and GCTST exhibited an osteoblast phenotype, expressing alkaline phosphatase, and the receptor activator for nuclear factor kappaB ligand (RANKL), a factor that is essential for osteoclast formation. These cells also expressed osteoprotegerin (OPG), an inhibitor of osteoclastogenesis, and TRAIL, a receptor that binds OPG. Lacunar resorption by giant cells isolated from GCTB and GCTST was inhibited by OPG, zoledronate, and calcitonin. These findings indicate that the mononuclear and giant-cell components of GCTB and GCTST have similar phenotypic features and that the accumulation of osteoclasts in these giant-cell-rich tumors occurs by a RANKL-dependent process. RANKL expression by osteoblastlike mononuclear stromal cells in these tumors stimulates osteoclast formation and resorption; this would account for the osteolysis associated with these giant-cell-rich tumors. Inhibitors of osteoclast formation and activity are likely to be effective in controlling the osteolysis associated with GCTB and possibly other giant-cell-rich lesions.

Topics: Apoptosis Regulatory Proteins; Bone Neoplasms; Bone Resorption; Carrier Proteins; Coculture Techniques; Giant Cell Tumor of Bone; Giant Cell Tumors; Glycoproteins; Humans; Immunohistochemistry; Membrane Glycoproteins; Monocytes; Osteoclasts; Osteolysis; Osteoprotegerin; Phenotype; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA, Messenger; Soft Tissue Neoplasms; Stromal Cells; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology, characterized by the presence of inflammatory synovitis accompanied by destruction of joint cartilage and bone. Treatment with vasoactive intestinal peptide (VIP) prevents experimental arthritis in animal models by downregulation of both autoimmune and inflammatory components of the disease. The aim of this study was to characterize the protective effect of VIP on bone erosion in collagen-induced arthritis (CIA) in mice. We have studied the expression of different mediators implicated in bone homeostasis, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), receptor activator of nuclear factor-kappaB (RANK), receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), IL-1, IL-4, IL-6, IL-10, IL-11 and IL-17. Circulating cytokine levels were assessed by ELISA and the local expression of mediators were determined by RT-PCR in mRNA extracts from joints. VIP treatment resulted in decreased levels of circulating IL-6, IL-1beta and TNFalpha, and increased levels of IL-4 and IL-10. CIA-mice treated with VIP presented a decrease in mRNA expression of IL-17, IL-11 in the joints. The ratio of RANKL to OPG decreased drastically in the joint after VIP treatment, which correlated with an increase in levels of circulating OPG in CIA mice treated with VIP. In addition, VIP treatment decreased the expression of mRNA for RANK, iNOS and COX-2. To investigate the molecular mechanisms involved, we tested the activity of NFkappaB and AP-1, two transcriptional factors closely related to joint erosion, by EMSA in synovial cells from CIA mice. VIP treatment in vivo was able to affect the transcriptional activity of both factors. Our data indicate that VIP is a viable candidate for the development of treatments for RA.

Topics: Animals; Arthritis, Experimental; Bone and Bones; Cyclooxygenase 2; Cytokines; Disease Models, Animal; Drug Evaluation; Gene Expression Profiling; Glycoproteins; I-kappa B Proteins; Inflammation Mediators; JNK Mitogen-Activated Protein Kinases; Male; Mice; Mice, Inbred DBA; NF-kappa B; NF-KappaB Inhibitor alpha; Nitric Oxide Synthase Type II; Osteoclasts; Osteolysis; Osteoprotegerin; Protein Transport; Proto-Oncogene Proteins c-jun; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA, Messenger; Transcription Factor AP-1; Transcription, Genetic; Vasoactive Intestinal Peptide

Periprosthetic osteolysis involves osteoclast activation by wear particulates and their exposure to mechanical perturbation through exposure to shear forces generated by periprosthetic fluid as well as interface micromotion. This study aimed to determine the interactions between wear particulates, mechanical stimulation, and osteoclasts. In static cultures, wear particulates increased osteoclast differentiation. Addition of neutralizing antibodies to RANKL (receptor activator of nuclear factor kappa ligand) inhibited the particle-induced increase in osteoclast numbers. Cyclic 5000 microstrains were applied with the use of a custom-built device to marrow-derived cultures to assess the effect on osteoclast differentiation. Mechanical strain application alone decreased osteoclast differentiation, which was further decreased by the addition of particles despite increases in the soluble RANKL to osteoprotegerin (OPG) ratio. Mechanical strain alone induced mature osteoclast apoptosis in a dose-dependent manner. In contrast, in the mature osteoclast model, the addition of nonmetal particulates protected the osteoclasts from becoming apoptopic. Titanium (Ti) and cobalt chromium (CoCr) particles, however, induced osteoclast apoptosis, whereas polyethylene (PE) and polymethylmethacrylate (PMMA) did not. Wear particulates and mechanical stimulation interact via an eicosanoid-dependent pathway to alter osteoclast function and survival. The addition of mechanical perturbation to a particle-laden system thus appears to enhance the potential for osteolytic activity by enhancing osteoclast survival.

Topics: Animals; Antibodies; Apoptosis; Bone Marrow; Carrier Proteins; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Chromium Alloys; Eicosanoids; Glycoproteins; Membrane Glycoproteins; Mice; Osteoclasts; Osteolysis; Osteoprotegerin; Particle Size; Plastics; Polyethylene; Rabbits; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Solubility; Stress, Mechanical; Surface Properties; Titanium

RANKL (receptor activator of nuclear factor kappaB ligand) promotes osteoclast differentiation, stimulates osteoclast activity, and prolongs osteoclast survival and adherence to bone. Abnormalities of the RANKL/RANK/osteoprotegerin system have been implicated in a range of diseases, including osteoporosis. To date, no work has been done in osteolytic lesions of the facial skeleton. In this study, specimens of ameloblastomas, dentigerous cysts, odontogenic keratocysts, and radicular cysts were subjected to immunohistochemical analysis for RANKL and tartrate-resistant acid phosphatase (TRAP). Immunofluorescence staining for TRAP was visualized under confocal microscopy. All specimens demonstrated distinct positive immunoreactivity to RANKL and TRAP. The TRAP-positive cells also stained with in situ hybridization for human calcitonin receptor, a definitive marker for osteoclasts. Mononuclear pre-osteoclasts were observed to migrate from blood to the connective tissue stroma and multinucleate toward the bone surface. It can be concluded that RANKL plays a role in bone resorption in osteolytic lesions of the facial skeleton.

Topics: Acid Phosphatase; Ameloblastoma; Dentigerous Cyst; Facial Bones; Glycoproteins; Humans; Immunohistochemistry; In Situ Hybridization; Isoenzymes; Jaw Neoplasms; Odontogenic Cysts; Osteolysis; Osteoprotegerin; Radicular Cyst; Receptors, Calcitonin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Signal Transduction; Tartrate-Resistant Acid Phosphatase

Multiple myeloma is a plasma cell malignancy characterized by the development of osteolytic lesions leading to bone pain, pathologic fractures, and hypercalcemia. Osteoprotegerin (OPG) is a potent inhibitor of osteoclast differentiation and activation, but is limited as a therapeutic agent due to its short circulating half-life. In order to overcome these limitations, the therapeutic effects of native OPG gene transfer are examined.. We used replication-incompetent lentiviral vectors to transfer the unmodified, native human OPG gene ex vivo into human ARH-77 cells injected into severe combined immunodeficient (SCID) mice, to determine gene transfer efficiency as well as the impact on disease progression in this in vivo model.. We can efficiently transfer and express either the LacZ marker gene or the native human OPG gene into human ARH-77 cells. Moreover, transfer of the OPG gene into ARH-77 cells reduces the development of osteolytic bony lesions when these cells are injected into SCID mice, compared to mice injected with either unmodified ARH-77 cells or ARH-77 cells transduced with the OPG gene in the antisense orientation. This therapeutic effect was manifested as a reduction in vertebral compression deformities and in the number and size of long-bone osteolytic lesions on skeletal radiographs, as well as a decrease in osteoclast surface on histologic analysis.. A lentiviral vector can efficiently transfer the native human OPG gene to myeloma cells ex vivo and inhibit myeloma-induced bone destruction, thereby suggesting a therapeutic potential for unmodified, native OPG gene transfer for osteoclast-dependent skeletal disorders.

Topics: Animals; Bone Density; Cell Line, Tumor; DNA, Antisense; DNA, Recombinant; Genetic Therapy; Genetic Vectors; Glycoproteins; Lac Operon; Lentivirus; Mice; Multiple Myeloma; Neoplasm Transplantation; Osteoclasts; Osteolysis; Osteoprotegerin; Pilot Projects; Radiography; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Transduction, Genetic; Xenograft Model Antitumor Assays

Orthopedic implant failures are often associated with peri-implant osteolysis. Particles generated from the wear process have been suspected to play an important role in this situation. Indeed, the peri-implant osteolysis could be due to the presence of particles stimulating the osteoclastogenesis process. We hypothesize then that the presence of a low particle concentration positively influences osteoblasts to produce osteoclastogenesis factors. If true, this hypothesis would then support the idea that the particles could be at the origin of the process leading to implant loosening. To check the validity of this hypothesis, we quantified in vitro the production of different genes involved in the osteoclastogenesis process using primary isolated human osteoblasts treated or not with particles. Results showed that low concentrations of particles might have a stimulating effect on osteoblasts to produce osteoclastogenesis factors as demonstrated by the increase of RANKL and CSF-1 gene expression in the particle group.

Topics: Biocompatible Materials; Carrier Proteins; Cells, Cultured; DNA Primers; Drug Delivery Systems; Gene Expression Regulation; Glycoproteins; Humans; Macrophage Colony-Stimulating Factor; Male; Membrane Glycoproteins; Middle Aged; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; Prosthesis Design; Prosthesis Failure; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA; Time Factors

Effective treatment for bacteria-induced bone lytic diseases is not yet available. In this study, we showed that PAR, an NF-kappaB inhibitor found in medicinal herbs, can block LPS-induced osteolysis. PAR does this by inhibiting osteoclastogenesis and bone resorption and promoting apoptosis of osteoclasts through the suppression of NF-kappaB activity.. Osteolysis induced by chronic gram-negative bacterial infection underlies many bone diseases such as osteomyelitis, septic arthritis, and periodontitis. Drugs that inhibit lipopolysaccharide (LPS)-induced osteolysis are critically needed for the prevention of bone destruction in infective bone diseases. In this study, we investigated the effect of parthenolide (PAR) on LPS-induced osteolysis in vivo and studied its role in osteoclastogenesis, bone resorption, apoptosis, and NF-kappaB activity.. The LPS-induced osteolysis in the mouse calvarium model was used to examine the effect of PAR in vivo. RANKL-induced osteoclast differentiation from RAW264.7 cells and bone resorption assays were used to assess the effect of PAR in vitro. Assays for NF-kappaB activation, p65 translocation, and IkappaB-alpha degradation were used to determine the mechanism of action of PAR in osteoclasts and their precursors. Flow cytometry and confocal microscopic analysis were used to examine cell apoptosis. Semiquantitative RT-PCR was performed to examine the effect of PAR on gene expression of RANK and TRAF6.. We found that PAR (0.5 and 1 mg/kg), injected simultaneously with LPS (25 mg/kg) or 3 days later, blocked the LPS-induced osteolysis in the mouse calvarium model. In vitro studies showed that low concentrations of PAR (<1 microM) inhibited in vitro osteoclastogenesis and osteoclastic bone resorption, whereas higher concentrations (>5 microM) triggered apoptotic cell death of osteoclasts and their precursor cells in a dose-dependent manner. Furthermore, PAR inhibited LPS-induced NF-kappaB activation, p65 translocation, and IkappaB-alpha degradation both in mature osteoclasts and their precursors in a time- and dose-dependent manner. In addition, PAR inhibited NF-kappaB activation induced by osteoclastogenic factors RANKL, interleukin (IL)-1beta, or TNF-alpha to varying degrees and reduced the gene expression of RANK and TRAF6.. The NF-kappaB pathway is known to mediate both osteoclast differentiation and survival. These findings indicate that PAR blocks LPS-induced osteolysis through the suppression of NF-kappaB activity and suggest that it might have therapeutic value in bacteria-induced bone destruction.

Topics: Active Transport, Cell Nucleus; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Blotting, Western; Bone and Bones; Bone Resorption; Cell Differentiation; Dose-Response Relationship, Drug; Flow Cytometry; Gene Expression Regulation; Genes, Reporter; Glycoproteins; Green Fluorescent Proteins; Interleukin-1; Lactones; Lipopolysaccharides; Luciferases; Macrophages; Mice; Mice, Inbred C57BL; Microscopy, Confocal; NF-kappa B; Osteoclasts; Osteolysis; Osteoprotegerin; Protein Transport; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; Sesquiterpenes; Time Factors; TNF Receptor-Associated Factor 6; Transcription, Genetic; Tumor Necrosis Factor-alpha

Prostate adenocarcinoma is associated with the formation of osteoblastic metastases in bone. It is hypothesized that osteoclastogenesis is a critical component in the development of skeletal metastases. These findings, however, were generally noted in predominantly osteolytic lesions. The pathophysiology of osteoblastic lesions remains unknown but the type of bone lesion formed may be influenced by the cytokines produced by prostate tumors. To test this theory, we implanted PC-3 and LAPC-9 cells into the tibias of SCID mice. These mice were sacrificed at 1, 2, 4, 6, and 8 weeks after implantation and histologic analysis was performed on these tibias. PCR analysis was also performed on bulk tumors. The results showed that the PC-3 implanted tibias developed pure osteolytic lesions while the LAPC-9 implanted tibias developed pure osteoblastic lesions on radiographs. Analysis of tibias after injection with PC-3 cells revealed progressive osteolytic lesions with abundant osteoclast activity at 2 weeks and destruction of the proximal tibia at 6 weeks after cell implantation. In contrast, the LAPC-9 cells formed osteoblastic lesions six weeks after cell injection. There were rare osteoclasts prior to the establishment of the osteoblastic lesions but greater osteoclast activity was noted with remodeling of the osteoblastic lesion 8 weeks after implantation of the tumor cells. PCR analysis revealed that PC-3 cells produced RANKL, IL-1, and TNF-alpha, which are associated with osteoclastogenesis. In contrast, LAPC-9 cells produced osteoprotegerin, which blocks osteoclast production and no detectable levels of RANKL or IL-1 and only minimal amounts of TNF-alpha were noted. These cells secreted BMP-2, -4, -6, and IL-6, which are associated with bone formation. These results suggest that the role of the osteoclast in the development of a metastatic lesion is variable depending on the phenotype of the prostate cancer cells, and that tumor-induced osteolysis may not be required for osteoblastic metastases.

Topics: Animals; Cytokines; Gene Expression; Glycoproteins; Humans; Immunohistochemistry; Male; Mice; Mice, SCID; Neoplasm Transplantation; Osteoblasts; Osteolysis; Osteoprotegerin; Phenotype; Polymerase Chain Reaction; Prostatic Neoplasms; Radiography; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tibia; Tumor Cells, Cultured

TNFalpha and IL-1alpha are proinflammatory cytokines that are abundant in periprosthetic tissues. These cytokines stimulate bone resorption and have recently been shown to directly induce osteoclast formation in mouse marrow cultures. We examined whether TNFalpha and IL-1alpha can directly induce osteoclast formation from human arthroplasty-derived (CD14(+)) macrophages by a mechanism independent of RANKL-induced osteoclastogenesis.. TNFalpha and M-CSF (+/-IL-1alpha) were added to cultures of magnetically sorted (CD14(+)) and unsorted (CD14(+)/CD14(-)) cells isolated from the pseudomembrane of loosened hip arthroplasties. Osteoprotegerin (OPG), RANK:Fc and antibodies to TNF receptors (p55 and p75) were added to these cultures to distinguish the pathway of osteoclastogenesis. Osteoclast differentiation was assessed by expression of tartrate-resistant acid phosphatase (TRAP), vitronectin receptor (VNR) and lacunar resorption.. The addition of TNFalpha (+/-IL-1alpha) resulted in differentiation of CD14(+) macrophages into TRAP(+) and VNR(+) multinucleated cells capable of extensive lacunar resorption. Both OPG and RANK:Fc (which inhibit RANKL-induced osteoclastogenesis) did not block osteoclastogenesis. The addition of antibodies directed against the p55 receptor subunit of TNF resulted in significant inhibition of osteoclast formation and lacunar resorption.. Our results indicate that, in the presence of M-CSF, TNFalpha is sufficient for inducing human osteoclast differentiation from arthroplasty macrophages and that TNFalpha acts synergistically with IL-1alpha to stimulate lacunar resorption. This process is distinct from the RANK/RANKL signalling pathway and is likely to operate in periprosthetic tissues when there is heavy wear particle deposition and cytokine production.

Topics: Acetabulum; Aged; Aged, 80 and over; Antibodies; Antigens, CD; Antineoplastic Agents; Bone Resorption; Cells, Cultured; Female; Fibroblasts; Glycoproteins; Humans; Lipopolysaccharide Receptors; Macrophages; Male; Middle Aged; Osteoclasts; Osteolysis; Osteoprotegerin; Prosthesis Failure; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; Signal Transduction; T-Lymphocytes; Tumor Necrosis Factor-alpha

Topics: Animals; Bone Neoplasms; Carrier Proteins; Glycoproteins; Humans; Joint Prosthesis; Membrane Glycoproteins; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Stromal Cells

The bone destruction in myeloma patients is largely responsible for the clinical features of the disease. However, only recently has attention focused on identifying and developing drugs targeted specifically at the osteolysis. Receptor activator of NF-kappaB ligand (RANKL), macrophage inflammatory protein (MIP)-1alpha, and proteasomal function have been implicated in the pathogenesis of myeloma and associated bone disease. We provide "proof of principle" in preclinical myeloma models that these are indeed valid molecular targets in development of novel therapeutics.. The efficacy of antagonists of RANKL and MIP-1alpha bioactivities (RANK.Fc and neutralizing monoclonal anti-MIP-1alpha antibody) in ameliorating osteolysis and reducing tumor burden was evaluated in a mouse model in which murine myeloma 5TGM1 cells are injected intravenously into syngeneic mice. In addition, the activity of a petidyl aldehyde proteasome inhibitor (proteasome inhibitor-1 [PSI]) on tumor growth was tested in a murine 5TGM1 plasmacytoma model and in mice intravenously inoculated with 5TGM1 cells.. RANK.Fc and anti-MIP-1alpha antibody inhibited the development and progression of osteolytic lesions and significantly reduced tumor load assessed by serum monoclonal paraprotein titers. Intratumoral injections of PSI inhibited growth of 5TGM1 plasmacytomas and induced tumor regression in some cases. In addition, systemic administration of PSI significantly prolonged time to onset of paraplegia in tumor-bearing mice.. The results highlight the critical roles of RANKL and MIP-1alpha in the development and progression of myeloma and provide a basis for future evaluation in myeloma patients of novel therapeutics that disrupt interactions of RANKL and MIP-1alpha with their cognate receptors. The data also suggest that further studies in preclincal myeloma models aimed at identifying other proteasome inhibitors with antitumor efficacy would be worthwhile.

Topics: Animals; Antibodies, Monoclonal; Boronic Acids; Bortezomib; Carrier Proteins; Chemokine CCL3; Chemokine CCL4; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Disease Models, Animal; Glycoproteins; Macrophage Inflammatory Proteins; Membrane Glycoproteins; Mice; Multienzyme Complexes; Multiple Myeloma; Osteolysis; Osteoprotegerin; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteins; Pyrazines; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

A major clinical feature in multiple myeloma is the development of osteolytic bone disease. The increase in bone destruction is due to uncontrolled osteoclastic bone resorption. Until recently the factors responsible for mediating the increase in osteoclast formation in myeloma have been unclear. However, recent studies have implicated a number of factors, including the ligand for receptor activator of NFkappaB (RANKL) and macrophage inflammatory protein-1alpha. The demonstration that increased osteoclastic activity plays a central role in this process and the identification of molecules that may play a critical role in the development of myeloma bone disease have resulted in studies aimed at identifying new approaches to treating this aspect of myeloma.. Studies have been performed to determine the ability of recombinant osteoprotegerin (Fc.OPG), a soluble decoy receptor for RANKL, and potent new bisphosphonates to inhibit the development of myeloma bone disease in the 5T2MM murine model of multiple myeloma.. Fc.OPG was shown to prevent the development of osteolytic bone lesions in 5T2MM bearing animals. These changes were associated with a preservation of the cancellous bone loss induced by myeloma cells and an inhibition of osteoclast formation. Bisphosphonates, including ibandronate and zoledronic acid, were also shown to inhibit the development of osteolytic bone lesions in the 5T2MM model and alternative models of myeloma bone disease.. Bisphosphonates and Fc.OPG are effective inhibitors of the development of osteolytic bone lesions in pre-clinical murine models of myeloma bone disease.

Topics: Animals; Diphosphonates; Disease Models, Animal; Glycoproteins; Mice; Mice, SCID; Multiple Myeloma; Osteoclasts; Osteolysis; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Familial expansile osteolysis (FEO) is a rare autosomal dominant disorder characterized by striking focal expansile osteolytic bone lesions and generalized osteopenia, often accompanied by characteristic early hearing loss and dental disease. The TNFRSF11A gene encodes the receptor activator of nuclear factor-kappaB (RANK), which has been demonstrated to be essential in bone remodeling and osteoclast differentiation. Identical insertional mutations in the first exon of RANK have been identified in all published FEO kindreds. The mutation is an 18 base pair tandem duplication in the sequence coding for the signal peptide of RANK, which causes an increase in NF-kappaB signaling. We report the identification and mutational analysis of two unrelated FEO patients. One had no family history of FEO, but presented with bilateral hearing loss at an early age, deterioration of teeth, and severe pain and swelling in the distal tibia before the age of 20. The second patient had a family history of FEO and exhibited an extensive expansile tibial lesion and lesions in one humerus and a phalanx. She also had early hearing loss and dental disease. Mutational analysis of the TNFRSF11A gene in our patients demonstrated an 18 base pair tandem duplication, one base proximal to the duplications previously reported. This novel mutation results in addition of the same six amino acids to the RANK signal peptide that has been observed previously. Further analysis of the exon 1 sequence demonstrated that it has the ability to form a stable secondary structure that may facilitate the generation of tandem duplications.

Topics: Aged; Amino Acid Sequence; Amino Acids; Base Sequence; Bone and Bones; DNA Mutational Analysis; Exons; Female; Gene Duplication; Glycoproteins; Hearing Loss; Humans; Molecular Sequence Data; Mutation; NF-kappa B; Osteolysis; Osteoprotegerin; Protein Sorting Signals; Protein Structure, Secondary; Radiography; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Signal Transduction

Interleukin 8 (IL-8) is a member of the alpha chemokine family of cytokines originally identified as a neutrophil chemoattractant. Recently, we reported that elevated levels of IL-8, but not parathyroid hormone-related protein (PTHrP), correlated with increased bone metastasis in a population of human breast cancer cells. We hypothesized that IL-8 expression by breast cancer cells would either indirectly influence osteoclastogenesis via nearby stromal cells or directly influence osteoclast differentiation and activity. In the present study, we investigated the role of IL-8 in the process of osteoclast formation and bone resorption, which is associated with metastatic breast cancer. The addition of recombinant human (rh) IL-8 (10 ng/ml) to cultures of stromal osteoblastic cells stimulated both RANKL mRNA expression and protein production, with no effect on the expression of osteoprotegerin. In addition, rhIL-8 also directly stimulated the differentiation of human peripheral blood mononuclear cells into bone-resorbing osteoclasts. In these cultures, IL-8 was able to stimulate human osteoclast formation even in the presence of excess (200 ng/ml) RANK-Fc. The effect of IL-8 on osteoclasts and their progenitors was associated with the cell surface expression of the IL-8-specific receptor (CXCR1) on the cells. These results demonstrate a direct effect of IL-8 on osteoclast differentiation and activity. Together, these data implicate IL-8 in the osteolysis associated with metastatic breast cancer.

Topics: Animals; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cell Line, Tumor; Glycoproteins; Humans; Interleukin-8; Mice; Osteoclasts; Osteolysis; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Proteins

Pathological osteolyses are considered a consequence of a disturbance in the mechanisms that govern the bone remodeling, mainly the communication between osteoclasts and osteoblasts. Osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) are newly discovered molecules that play a key role in these communications. RANKL is essential for osteoclast differentiation via its receptor RANK located on the osteoclast membrane. OPG is a soluble decoy receptor that inhibits osteoclast differentiation through its binding to RANKL. The aim of this study is the analysis of the RANKL/OPG balance by complementary methods (semiquantitative reverse transcription-polymerase chain reaction, immunohistochemistry, and enzyme-linked immunosorbent assay) in human osteolysis associated to various bone etiologies (n = 60), tumoral (primitive, secondary) or not, compared to healthy tissues (n = 16). Results demonstrated that RANKL/OPG ratio was significantly increased in patients suffering from severe osteolysis compared to the control group and that this imbalance is involved in bone resorption mechanisms. In this study, OPG expression appears to reflect a protective mechanism of the skeleton to compensate increased bone resorption by inhibiting osteoclast formation and bone resorbing activity. Moreover, as revealed by immunohistochemistry, RANKL and OPG were colocalized in all of the tissues analyzed. To define the veracity of RANKL/OPG index in assessing and managing patients with severe osteolysis, an extended population of patients suffering from severe osteolysis must be now monitored.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Carrier Proteins; Enzyme-Linked Immunosorbent Assay; Female; Glycoproteins; Humans; Immunohistochemistry; Male; Membrane Glycoproteins; Middle Aged; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The pathogenesis of periprosthetic bone loss in aseptic and septic prosthesis loosening is unclear. There is considerable evidence that macrophages and osteoclasts play a key role in focal bone erosion and osteolysis around the prosthesis. RANKL (receptor activator of nuclear factor kappaB ligand) was shown to be a potent osteoclastogenic factor, and to be involved in bone destruction of myeloma and rheumatoid arthritis patients. Osteoprotegerin (OPG) is the natural RANKL inhibitor and may prevent periprosthetic bone loss.. The presence and distribution of RANKL, its receptor RANK and OPG in the periprosthetic interface of septically (n = 5) and aseptically (n = 6) loosened prostheses was examined by immunohistochemistry and immunoblotting. Additionally, the immunophenotype of the inflammatory infiltrate was determined [CD3, CD68, Ki-67, tartrate-resistant acid posphatase (TRAP)].. Aseptic and septic cases revealed a different histopathologic pattern. However, in all cases RANKL and RANK could be demonstrated in macrophages and giant cells. In addition, RANKL detected by immunoblot analysis proved to have the same molecular weight as a recombinant RANKL used as a control (31 kD and approximately 48 kD). OPG was detected in aseptic loosening, where macrophages showed a strong staining, but multinucleated giant cells were only weakly stained. A weak OPG staining was also observed in septic loosening.. The pathogenesis of bone loss in septic loosening remains unclear, because the septic membrane bears few macrophages and giant cells, and half of them express OPG. In aseptic loosening, macrophages might not be stimulated by RANKL as a result of OPG expression. But multinucleated giant cells may be activated, as they hardly express OPG. They might be responsible for periprosthetic bone loss in aseptic loosening as a result of their RANKL and RANK expression.

Topics: Aged; Aged, 80 and over; Arthroplasty, Replacement, Hip; Arthroplasty, Replacement, Knee; Blotting, Western; Carrier Proteins; Female; Giant Cells; Glycoproteins; Hip Prosthesis; Humans; Immunohistochemistry; Knee Prosthesis; Macrophages; Male; Membrane Glycoproteins; Middle Aged; Osteolysis; Osteoprotegerin; Prosthesis Failure; Prosthesis-Related Infections; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Aseptic loosening of orthopaedic implants secondary to wear debris-induced osteolysis is a serious problem. Osteoprotegerin (OPG) is a natural decoy protein that inhibits osteoclast activation and bone resorption. This study investigated whether gene therapy using a recombinant adeno-associated viral vector that expresses OPG can inhibit wear debris-induced osteolysis.. A recombinant adeno-associated virus (rAAV) vector co-expressing OPG (rAAV-OPG-IRES-EGFP) was generated. A control vector expressing b-galactosidase (rAAV-LacZ) was also prepared. In vitro validation experiments were performed to determine rAAV-OPG-IRES-EGFP transduction efficiency, OPG expression level and function in bone wafer, and osteoclastic activity. The effect of rAAV-OPG-IRES-EGFP in vivo gene therapy on wear debris-induced osteolysis was then evaluated in a mouse calvarial model in which a single intramuscular injection of the vector was administered prior to the introduction of the wear debris. The effects of the rAAV-OPG-IRES-EGFP gene therapy on wear debris-induced osteoclastogenesis and bone resorption were determined by histomorphometry on day 10.. In vitro experiments revealed that 100% of human embryonic kidney 293 cells were transduced at a multiplicity of infection of 1000 with both rAAV-OPG-IRES-EGFP and rAAV-LacZ. At a rAAV-OPG-IRES-EGFP multiplicity of infection of 1000, an OPG concentration of 135 ng/mL of culture media was achieved after four days. Using a bone-wafer assay for osteoclast activity, we found that treatment with rAAV-OPG-IRES-EGFP reduced resorption sevenfold compared with parathyroid hormone-stimulated controls and elevenfold compared with rAAV-LacZ controls. Furthermore, a seventeenfold decrease in RANKL and macrophage colony-stimulating factor-induced splenocyte osteoclastogenesis was observed in co-cultures containing rAAV-OPG-IRES-EGFP-infected fibroblasts. In vivo administration of rAAV-OPG-IRES-EGFP resulted in detectable transduction of myocytes at the injection site and a significant increase in expression of serum OPG levels by the second day (p < 0.05). Maximal concentrations were obtained on day 6 and then leveled off throughout the observation period. In contrast, serum OPG could not be detected in the sham-treated, uninfected titanium-stimulated, or rAAV-LacZ-infected mice. In the control mice, titanium implantation resulted in a threefold increase in the mean number of osteoclasts adjacent to the sagittal suture as well as a twofold increase in the mean area of soft tissue in the sagittal suture compared with the sham-treated mice. In contrast, osteoclast numbers remained at basal levels, and the area of soft tissue in the sagittal suture was markedly reduced in titanium-implanted animals that received rAAV-OPG-IRES-EGFP treatment, demonstrating a complete inhibition of osteolysis in response to titanium particles.. A single intramuscular injection of the rAAV-OPG-IRES-EGFP vector can efficiently transduce myocytes to produce high levels of OPG. The OPG effectively inhibits wear debris-induced osteoclastogenesis and osteolysis.. Currently, there is no approved drug therapy to prevent or inhibit periprosthetic osteolysis. Although preclinical studies have identified potential drug therapies (i.e., bisphosphonates), there is no evidence that these drugs can effectively treat aseptic loosening in patients. This is the first evidence that in vivo OPG gene therapy can be used to prevent wear debris-induced osteolysis.

Topics: Animals; Dependovirus; Genetic Therapy; Genetic Vectors; Glycoproteins; Humans; Male; Mice; Mice, Inbred C57BL; Osteolysis; Osteoprotegerin; Prosthesis Failure; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Transduction, Genetic

Osteoprotegerin (OPG), a natural negative regulator of osteoclastogenesis and bone resorption, may be a potential therapeutic agent for treatment of osteolysis-associated prosthetic joint loosening. Using an in vivo adeno-associated virus (AAV)-mediated gene transfer technique, this study was designed to evaluate the protective effects of OPG transgene against orthopedic wear debris-induced bone loss in a murine model of osteolysis.. Bone tissue was implanted into established pouches on BALB/c mice, followed by the introduction of ultra-high-molecular-weight polyethylene (UHMWPE) particles to provoke inflammation and osteolysis. The viruses encoding human OPG gene (rAAV-hOPG) or beta-galactosidase marker gene (rAAV-LacZ) were injected into the air pouches, and the tissue was harvested 7 days after viral infection for histologic and molecular analyses.. Successful transgene expression was confirmed by the detection of OPG by enzyme-linked immunosorbent assay and positive X-Gal staining of pouch tissue (LacZ). Real-time polymerase chain reaction indicated significant diminishment of messenger RNA expression of osteoclast markers in OPG-transduced pouches compared with rAAV-LacZ-transduced pouches. The transduction and expression of OPG also markedly decreased the gene copies of the biologic receptor activator of nuclear factor kappaB. The expression of OPG in the bone-implanted pouch reduced bone calcium release by a mean of 39% compared with the calcium release in the other 2 groups. Computerized image analysis revealed that expression of OPG significantly protected against bone collagen loss.. OPG gene transfer mediated by rAAV effectively protects against particulate polyethylene-induced bone resorption in this experimental model. Data suggest that gene transfer using rAAV-OPG may be a feasible and effective therapeutic candidate to treat or prevent wear debris-associated osteolysis and aseptic loosening.

Topics: Animals; Cell Division; Dependovirus; Gene Expression; Gene Transfer Techniques; Genetic Vectors; Glycoproteins; Humans; Inflammation; Mice; Mice, Inbred BALB C; Osteoclasts; Osteolysis; Osteoprotegerin; Polyethylene; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Transduction, Genetic

Topics: Adolescent; Adult; Age of Onset; Bone Diseases, Metabolic; Child; Female; Glycoproteins; Humans; Male; Mutagenesis, Insertional; Osteolysis; Osteoprotegerin; Radiography; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Spain

The biologic mechanisms involved in the pathogenesis of multiple myeloma (MM) bone disease are not completely understood. Recent evidence suggests that T cells may regulate bone resorption through the cross-talk between the critical osteoclastogenetic factor, receptor activator of nuclear factor-kappaB ligand (RANKL), and interferon gamma (IFN-gamma) that strongly suppresses osteoclastogenesis. Using a coculture transwell system we found that human myeloma cell lines (HMCLs) increased the expression and secretion of RANKL in activated T lymphocytes and similarly purified MM cells stimulated RANKL production in autologous T lymphocytes. In addition, either anti-interleukin 6 (anti-IL-6) or anti-IL-7 antibody inhibited HMCL-induced RANKL overexpression. Consistently, we demonstrated that HMCLs and fresh MM cells express IL-7 mRNA and secrete IL-7 in the presence of IL-6 and that bone marrow (BM) IL-7 levels were significantly higher in patients with MM. Moreover, we found that the release of IFN-gamma by T lymphocytes was reduced in presence of both HMCLs and purified MM cells. Furthermore, in a stromal cell-free system, osteoclastogenesis was stimulated by conditioned medium of T cells cocultured with HMCLs and inhibited by recombinant human osteoprotegerin (OPG; 100 ng/mL to 1 microg/mL). Finally, RANKL mRNA was up-regulated in BM T lymphocytes of MM patients with severe osteolytic lesions, suggesting that T cells could be involved at least in part in MM-induced osteolysis through the RANKL overexpression.

Topics: Aged; Bone Marrow Cells; Carrier Proteins; Coculture Techniques; Culture Media, Conditioned; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Interleukin-6; Interleukin-7; Lymphocyte Activation; Male; Membrane Glycoproteins; Middle Aged; Multiple Myeloma; Neoplasm Proteins; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Stromal Cells; T-Lymphocytes

On June 26-27, 2001, the Sixth Research Roundtable in Multiple Myeloma, entitled "The Role of the Bone Microenvironment in Multiple Myeloma," was held and focused on the biology of cell-to-cell interactions, the mediators of bone disease, and novel treatment strategies for myeloma. Studies on cell-cell interactions showed that vascular cell adhesion molecule 1, expressed by local endothelial and stromal cells, binds to tumor cell surface integrins in which expression may be increased by tumor cell-derived chemokines such as macrophage inflammatory protein (MIP) 1alpha. These adhesive interactions increase production and release of vascular endothelial growth factor (VEGF). Studies on myeloma bone disease showed the ligand for receptor activator of nuclear transcription factor-kappaB (RANKL) was expressed on tumor cells and stromal cells associated with myeloma cells and was critical for osteoclast-induced osteolysis. Blockade of RANKL suppressed osteoclast maturation, bone resorption, and tumor development. Bisphosphonates, in addition to reducing osteoclast mobility and inducing osteoclast apoptosis, also decreased tumor cell adhesion to stroma. Immunomodulatory drugs such as thalidomide analogues targeted these tumor cell-stromal cell interactions, blocking both secretion of cytokines and activation of intracellular signaling pathways required for tumor survival and growth. These agents induced tumor cell apoptosis, decreased neovascularization, and potentiated natural killer cell activity. The proteasome inhibitor PS-341 also prevented expression of adhesion molecules and cytokines and triggered tumor cell apoptosis, even in drug-resistant cell lines, while showing minimal activity in healthy cells. In addition, potential therapeutic agents under investigation, which included RANKL antagonists, protein prenylation inhibitors, and osteoblast growth factors, were discussed.

Topics: Adjuvants, Immunologic; Bone and Bones; Bone Marrow; Bone Resorption; Boronic Acids; Bortezomib; Carrier Proteins; Cell Division; Chemokine CCL3; Chemokine CCL4; Diphosphonates; Endothelial Growth Factors; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Lymphokines; Macrophage Inflammatory Proteins; Membrane Glycoproteins; Multiple Myeloma; Osteoclasts; Osteolysis; Osteoprotegerin; Protease Inhibitors; Protein Prenylation; Pyrazines; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Expansile skeletal hyperphosphatasia (ESH) is a singular disorder characterized in the year 2000 in a mother and daughter with early-onset deafness, premature loss of teeth, progressive hyperostotic widening of long bones causing painful phalanges in the hands, accelerated bone remodeling, and episodic hypercalcemia likely inherited as a highly penetrant, autosomal dominant trait. Absence of large osteolytic lesions with cortical thinning in major long bones, together with bouts of hypercalcemia, indicated that ESH is not a variant of familial expansile osteolysis (FEO). Here, we investigated the molecular basis of ESH after three families with FEO were reported to have an identical 18-base pair tandem duplication (84dup18) in the signal peptide sequence of the TNFRSF11A gene that encodes receptor activator of nuclear factor-kappaB (RANK). We find that ESH is caused by a remarkably similar 15-base pair tandem duplication (84dup15) in TNFRSF11A. Hence, ESH and FEO are allelic diseases and ESH, like FEO, probably reflects increased activity in the skeleton of the RANK target, nuclear factor-kappaB (NF-kappaB).

Topics: Alkaline Phosphatase; Alleles; Amino Acid Sequence; Base Sequence; Bone Diseases, Metabolic; DNA; Female; Genes, Dominant; Glycoproteins; Humans; Molecular Sequence Data; NF-kappa B; Osteolysis; Osteoprotegerin; Phosphorus Metabolism Disorders; Protein Sorting Signals; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tandem Repeat Sequences

Prosthesis failure due to wear debris-induced osteolysis remains a major clinical problem and the greatest limitation for total joint arthroplasty. Based on our knowledge of osteoclast involvement in this process and the requirements of receptor activator of NF-kappaB (RANK) signaling in osteoclastogenesis and bone resorption, we investigated the efficacy of RANK blockade in preventing and ameliorating titanium (Ti)-induced osteolysis in a mouse calvaria model. Compared with placebo controls we found that all doses of RANK:Fc above 1 mg/kg intraperitoneally (ip) per 48 h significantly inhibited osteoclastogenesis and bone resorption in response to Ti implanted locally. Complete inhibition occurred at 10 mg/kg ip per 48 h, yielding results that were statistically equivalent to data obtained with Ti-treated RANK-/- mice. We also evaluated the effects of a single injection of RANK:Fc on day 5 on established osteolysis and found that Ti-treated were still depleted for multinucleated tartrate-resistant acid phosphatase-positive (TRAP+) cells 16 days later. More importantly, this osteoclast depletion did not affect bone formation because the bone lost from the osteolysis on day 5 was restored by day 21. An assessment of the quantity and quality of the newly formed bone in these calvariae by calcein labeling and infrared (IR) microscopy, respectively, showed no significant negative effect of RANK:Fc treatment. These studies indicate that osteoclast depletion via RANK blockade is an effective method to prevent and reverse wear debris-induced osteolysis without jeopardizing osteogenesis.

Topics: Acid Phosphatase; Animals; Bone Matrix; Bone Remodeling; Bone Resorption; Female; Fluoresceins; Glycoproteins; Mice; Mice, Inbred CBA; Osteoclasts; Osteogenesis; Osteolysis; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Signal Transduction; Skull; Titanium

Aseptic loosening of prosthetic implants remains a serious orthopaedic problem and the greatest limitation to total joint arthroplasty. Central to the etiology of aseptic loosening is periprosthetic osteolysis at the bone-implant interface, which is caused by wear debris-induced inflammation. This inflammation produces the critical osteoclast differentiation factor RANKL, which directly stimulates osteoclastogenesis and osteoclastic bone resorption. A dominant factor known to counteract this process is the natural RANKL receptor antagonist protein OPG. Here we explore the potential of ex vivo OPG gene therapy for aseptic loosening by evaluating the eflicacy of stably transfected fibroblast-like synoviocytes (FLS) expressing OPG in preventing wear debris-induced osteoclastogenesis, in a mouse calvaria model. Although the stably transfected fibroblasts produced small amounts of OPG (0.3 ng/ml/72 h/10(6) cells), this protein was very effective in preventing osteoclastic resorption as determined in a bone wafer assay. More importantly. implantation of 10(7) FLS-OPG, together with 30 mg of Ti wear debris, onto the calvaria of mice, completely inhibited osteoclastogenesis 3 days after surgery. Animals given FLS-LacZ control cells, which persisted for 3 days as determined by X-gal staining, together with the Ti particles, had a 6-fold increase in osteoclastogenesis compared to controls without Ti. This increased osteoclastogenesis was completely inhibited by the FLS-OPG, as osteoclast numbers in the calvaria of these animals were similar to that seen in the SHAM controls.

Topics: Animals; Animals, Newborn; beta-Galactosidase; Cells, Cultured; Culture Media, Conditioned; Disease Models, Animal; Female; Fibroblasts; Genetic Therapy; Glycoproteins; Lac Operon; Mice; Mice, Inbred CBA; Osteoclasts; Osteolysis; Osteoprotegerin; Prostheses and Implants; Prosthesis Failure; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Skull; Titanium; Transduction, Genetic

We previously reported that mouse mammary carcinoma cell lines (MMT060562 and BALB/c-MC) induced osteoclast formation through production of prostaglandin E2 (PGE2) in cocultures with mouse bone marrow cells, but the mechanism(s) of PG production remained unclear. In the present in vitro and in vivo studies, we tested the involvement of cyclo-oxygenase-2 (COX-2), an inducible rate-limiting enzyme in PG biosynthesis, in the stimulation of osteoclast formation by mouse mammary carcinoma cell lines. Addition of a selective COX-2 inhibitor, JTE-522, to cocultures of mammary carcinoma cell lines and bone marrow cells lowered PGE2 concentration in the culture media and inhibited osteoclast formation in a dose-dependent manner. Northern blotting showed a very high level of COX-2 messenger RNA (mRNA) expression in MMT060562. The mRNA expression was low in BALB/c-MC, but it increased when BALB/c-MC and bone marrow cells were cocultured. The results of immunocytochemistry for COX-2 protein in respective cultures were compatible with the results of COX-2 mRNA. In vivo, BALB/c-MC injected into the heart of Balb/c mice metastasized to bone and formed osteolytic lesions in their hindlimbs. Histological examination revealed that tumor cells had metastasized to the bone marrow cavity and destroyed the bone trabeculae. Immunohistochemistry demonstrated that bone marrow stromal cells adjacent to tumor cells expressed COX-2 protein. These findings suggest that COX-2 plays an important role in the osteolysis of bone metastasis in vivo as well as in osteoclast formation in cocultures used as an in vitro model of metastatic bone disease.

Topics: Animals; Bone Neoplasms; Coculture Techniques; Cyclooxygenase 2; Dinoprostone; Female; Glycoproteins; Isoenzymes; Male; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Osteoclasts; Osteolysis; Osteoprotegerin; Prostaglandin-Endoperoxide Synthases; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

The osteoprotegerin-ligand (OPG-L) has been identified as the essential factor required for osteoclastogenesis, and its effects are prevented by the osteoprotegerin (OPG). The OPG-L/OPG balance plays a crucial role in coordinating the sequence of osteoclast and osteoblast differentiation during the bone remodeling. The aim of the study was to investigate if polymethylmethacrylate-based cements are able to modulate the expression of OPG-L/OPG in MG63 cells, which are known to have high levels of OPG and inducible expression of OPG-L. Four radio-opaque cements. namely Sulfix-60, CMW1, CMW2 and CMW3, were polymerized for either 1 h or 7 d. MG63 were incubated for 24 h with culture medium only, cement extracts and 2 microg/ml of human recombinant IL-1beta as positive control. An RT-PCR was performed to detect the OPG and OPG-L expression, and the house-keeping gene, GAPDH, was used as a reference for the semi-quantitative analysis. An increase in the OPG-L band density was observed for all cements, and consequently, the OPG-L/OPG ratio also increased. The ability of bone cements to induce the expression of OPG-L could be a co-factor in the development of osteolysis at the bone-cement interface.

Topics: Bone Cements; Carrier Proteins; Cell Line; Gene Expression; Glycoproteins; Humans; In Vitro Techniques; Materials Testing; Membrane Glycoproteins; Osteoblasts; Osteolysis; Osteoprotegerin; Polymethyl Methacrylate; Prosthesis Failure; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA, Messenger

Bone cancer pain most commonly occurs when tumors originating in breast, prostate, or lung metastasize to long bones, spinal vertebrae, and/or pelvis. Primary and metastatic cancers involving bone account for approximately 400,000 new cancer cases per year in the United States alone, and >70% of patients with advanced breast or prostate cancer have skeletal metastases. Whereas pain resulting from bone cancer can dramatically impact an individual's quality of life, very little is known about the mechanisms that generate and maintain this pain. To begin to define the mechanisms that give rise to advanced bone cancer pain, osteolytic 2472 sarcoma cells or media were injected into the intramedullary space of the femur of C3H/HeJ mice, and the injection hole was sealed using dental amalgam, confining the tumor cells to the bone. Twelve days after injection of 2472 tumor cells, animals showed advanced tumor-induced bone destruction of the injected femur, bone cancer pain, and a stereotypic set of neurochemical changes in the spinal cord dorsal horn that receives sensory inputs from the affected femur. Administration of osteoprotegerin, a naturally secreted decoy receptor that inhibits osteoclast maturation and activity and induces osteoclast apoptosis, or vehicle was begun at 12 days, when significant bone destruction had already occurred, and administration was continued daily until day 21. Ongoing pain behaviors, movement-evoked pain behaviors, and bone destruction were assessed on days 10, 12, 14, 17, and 21. The neurochemistry of the spinal cord was evaluated at days 12 and 21. Results indicated that osteoprotegerin treatment halted further bone destruction, reduced ongoing and movement-evoked pain, and reversed several aspects of the neurochemical reorganization of the spinal cord. Thus, even in advanced stages of bone cancer, ongoing osteoclast activity appears to be involved in the generation and maintenance of ongoing and movement-evoked pain. Blockade of ongoing osteoclast activity appears to have the potential to reduce bone cancer pain in patients with advanced tumor-induced bone destruction.

Topics: Animals; Bone Neoplasms; Disease Models, Animal; Glycoproteins; Male; Mice; Mice, Inbred C3H; Neurons, Afferent; Osteoclasts; Osteolysis; Osteoprotegerin; Pain; Proto-Oncogene Proteins c-fos; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Sarcoma, Experimental; Spinal Cord

Topics: Bone Neoplasms; Carrier Proteins; Glycoproteins; Humans; Male; Membrane Glycoproteins; Osteoclasts; Osteolysis; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Certain malignancies, including breast cancer, frequently metastasize to bone, where the tumor cells induce osteoclasts to locally destroy bone. Osteoprotegerin (OPG), a member of the tumor necrosis factor receptor family, is a negative regulator of osteoclast differentiation, activation, and survival. We tested the ability of recombinant OPG to inhibit tumor-induced osteoclastogenesis, osteolysis, and skeletal tumor burden in two animal models. In a syngeneic model, mouse colon adenocarcinoma (Colon-26) cells were injected into the left ventricle of mice. Treatment with OPG dose-dependently decreased the number and area of radiographically evident lytic bone lesions, which, at the highest dose, were undetectable. Histologically, OPG also decreased skeletal tumor burden and tumor-associated osteoclasts. In a nude mouse model, OPG treatment completely prevented radiographic osteolytic lesions caused by human MDA-MB-231 breast cancer cells. Histologically, OPG decreased skeletal tumor burden by 75% and completely eradicated MDA tumor-associated osteoclasts. In both models, OPG had no effect on metastatic tumor burden in a panel of soft tissue organs. These data indicate that OPG may be an effective therapy for preventing osteolysis and decreasing skeletal tumor burden in patients with bone metastasis.

Topics: Adenocarcinoma; Animals; Bone Neoplasms; Breast Neoplasms; Cell Line, Transformed; Colonic Neoplasms; Disease Models, Animal; Dose-Response Relationship, Drug; Glycoproteins; Humans; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Mice, Nude; Osteolysis; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Xenograft Model Antitumor Assays

Osteoprotegerin (OPG), the neutralizing decoy receptor for the osteoclast activator RANK ligand, was measured in serum taken from patients with multiple myeloma at the time of diagnosis. Median OPG was lower in the patients with myeloma (7.4 ng/mL; range, 2.6-80; n = 225) than in healthy age- and sex-matched controls (9.0 ng/mL; range 5.1-130; n = 40; P =.02). Importantly, OPG levels were associated with degree of radiographically assessed skeletal destruction (P =.01). The median OPG level in patients lacking osteolytic lesions was 9.1 ng/mL, as compared with 7.6 ng/mL and 7.0 ng/mL, respectively, in patients with minor or advanced osteolytic disease. Furthermore, OPG levels were associated with World Health Organization performance status (P =.003) and correlated to serum levels of carboxy-terminal propeptide of type I procollagen (PICP; P <.001) but not with clinical stage or survival. These findings suggest impaired OPG function in myeloma and give a rationale for OPG as a therapeutic agent against myeloma bone disease.

Topics: Adult; Aged; Aged, 80 and over; Case-Control Studies; Glycoproteins; Health Status Indicators; Humans; Middle Aged; Multiple Myeloma; Osteolysis; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Multiple myeloma is a B-cell malignancy characterized by the accumulation of plasma cells in the bone marrow and the development of osteolytic bone disease. The present study demonstrates that myeloma cells express the critical osteoclastogenic factor RANKL (the ligand for receptor activator of NF-kappa B). Injection of 5T2MM myeloma cells into C57BL/KaLwRij mice resulted in the development of bone disease characterized by a significant decrease in cancellous bone volume in the tibial and femoral metaphyses, an increase in osteoclast formation, and radiologic evidence of osteolytic bone lesions. Dual-energy x-ray absorptiometry demonstrated a decrease in bone mineral density (BMD) at each of these sites. Treatment of mice with established myeloma with recombinant osteoprotegerin (OPG) protein, the soluble decoy receptor for RANKL, prevented the development of lytic bone lesions. OPG treatment was associated with preservation of cancellous bone volume and inhibition of osteoclast formation. OPG also promoted an increase in femoral, tibial, and vertebral BMD. These data suggest that the RANKL/RANK/OPG system may play a critical role in the development of osteolytic bone disease in multiple myeloma and that targeting this system may have therapeutic potential.

Topics: Animals; Bone and Bones; Bone Density; Carrier Proteins; Flow Cytometry; Gene Expression; Glycoproteins; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Multiple Myeloma; Neoplasm Transplantation; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Topics: Bone Neoplasms; Chronic Disease; Glycoproteins; Models, Biological; Neural Conduction; Osteoclasts; Osteolysis; Osteoprotegerin; Pain; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Spinal Cord; Synaptic Transmission

Topics: Animals; Bone Neoplasms; Bone Resorption; Carrier Proteins; Glycoproteins; Membrane Glycoproteins; Mice; Osteoclasts; Osteolysis; Osteoprotegerin; Osteosarcoma; Pain; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Osteoprotegerin and osteoprotegerin ligand have recently been identified as novel proteins that inhibit and stimulate, respectively, osteoclast formation. We examined the possibility that osteoprotegerin would inhibit cancer-induced osteoclastogenesis and cancer growth in bone. An experimental model was used in which osteolytic tumors are known to stimulate osteoclastogenesis and grow in femora of osteoclast-deficient mice (op/op). Osteoprotegerin treatment decreased the number of osteoclasts by 90% (p < 0.0007) at sites of tumor in a dose-dependent manner and decreased bone tumor area by greater than 90% (p < 0.003). The mechanisms through which osteoprotegerin decreased osteoclast formation in tumor-bearing animals included (a) an osteoprotegerin-mediated, systemic reduction in the number of splenic and bone marrow-residing osteoclast precursor cells, (b) a decrease in the number of osteoclast precursor cells at sites of tumor as detected by cathepsin K and receptor activator of NFkappaB mRNA expression, and (c) a decrease in osteoprotegerin ligand mRNA at sites of tumor. These findings suggest that osteoprotegerin treatment, in addition to having direct antagonistic effects on endogenous osteoprotegerin ligand, decreases the number of osteoclast precursors and reduces production of osteoprotegerin ligand at sites of osteolytic tumor.

Topics: Animals; Bone Neoplasms; Carrier Proteins; Disease Models, Animal; Dose-Response Relationship, Drug; Femur; Gene Expression Regulation, Neoplastic; Glycoproteins; Macrophage Colony-Stimulating Factor; Membrane Glycoproteins; Mice; Mice, Inbred Strains; Osteoclasts; Osteolysis; Osteopetrosis; Osteoprotegerin; Parathyroid Hormone-Related Protein; Proteins; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA, Messenger; Stem Cells; Stromal Cells; Tumor Cells, Cultured

Breast cancers commonly cause osteolytic metastases in bone, a process that is dependent upon osteoclast-mediated bone resorption. Recently the osteoclast differentiation factor (ODF), better termed RANKL (receptor activator of NF-kappaB ligand), expressed by osteoblasts has been cloned as well as its cognate signaling receptor, receptor activator of NFkappaB (RANK), and a secreted decoy receptor osteoprotegerin (OPG) that limits RANKL's biological action. We determined that the breast cancer cell lines MDA-MB-231, MCF-7, and T47D as well as primary breast cancers do not express RANKL but express OPG and RANK. MCF-7, MDA-MB-231, and T47D cells did not act as surrogate osteoblasts to support osteoclast formation in coculture experiments, a result consistent with the fact that they do not express RANKL. When MCF-7 cells overexpressing PTH-related protein (PTHrP) were added to cocultures of murine osteoblasts and hematopoietic cells, osteoclast formation resulted without the addition of any osteotropic agents; cocultures with MCF-7 or MCF-7 cells transfected with pcDNAIneo required exogenous agents for osteoclast formation. When MCF-7 cells overexpressing PTHrP were cultured with murine osteoblasts, osteoblastic RANKL messenger RNA (mRNA) levels were enhanced and osteoblastic OPG mRNA levels diminished; MCF-7 parental cells had no effect on RANKL or OPG mRNA levels when cultured with osteoblastic cells. Using a murine model of breast cancer metastasis to bone, we established that MCF-7 cells that overexpress PTHrP caused significantly more bone metastases, which were associated with increased osteoclast formation, elevated plasma PTHrP concentrations and hypercalcaemia compared with parental or empty vector controls.

Topics: Animals; Carrier Proteins; Cell Division; Coculture Techniques; Female; Glycoproteins; Male; Mammary Neoplasms, Animal; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; Parathyroid Hormone-Related Protein; Proteins; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA, Messenger; Tumor Cells, Cultured