osteoprotegerin and Osteopetrosis

Osteoporosis is caused by imbalance between osteoclastic bone resorption and osteoblastic bone formation. From the recent results of several kinds of knockout mice, osteoclast differentiation factor (RANKL) and its soluble decoy receptor for RANKL (OPG) are essentially involved in pathogenesis of osteoporosis. Deficiency of RANKL in human has been shown to result in osteopetrosis. Furthermore, it has been reported that anti-RANKL neutralizing antibody (denosumab) will be effective new drug for osteoporosis.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bone Resorption; Cell Differentiation; Denosumab; Drug Design; Humans; Mice; Molecular Targeted Therapy; Mutation; Osteoblasts; Osteoclasts; Osteopetrosis; Osteoporosis; Osteoprotegerin; RANK Ligand; Signal Transduction

Topics: Adult; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bone and Bones; Denosumab; Homeostasis; Humans; Male; Mutation; Osteitis Deformans; Osteoclasts; Osteopetrosis; Osteoporosis; Osteoporosis, Postmenopausal; Osteoprotegerin; RANK Ligand

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

Topics: Animals; Bone and Bones; Bone Remodeling; Carrier Proteins; Glycoproteins; Humans; Membrane Glycoproteins; Models, Genetic; Osteoblasts; Osteoclasts; Osteopetrosis; Osteoporosis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

In 1997, investigators isolated a secreted glycoprotein that blocked osteoclast differentiation from precursor cells, prevented osteoporosis (decreased bone mass) when administered to ovariectomized rats, and resulted in osteopetrosis (increased bone mass) when overexpressed in transgenic mice. Since then, the isolation and characterization of the protein named osteoprotegerin (OPG) has stimulated much work in the fields of endocrinology, rheumatology, and immunology. OPG functions as a soluble decoy receptor for receptor activator of nuclear factor-kappaB ligand (RANKL, or OPG ligand) and shares homologies with other members of the tumor necrosis factor receptor superfamily. OPG acts by competing with the receptor activator of nuclear factor-kappaB, which is expressed on osteoclasts and dendritic cells for specifically binding to RANKL. RANKL is crucially involved in osteoclast functions and bone remodeling as well as immune cell cross-talks, dendritic cell survival, and lymph node organogenesis. More recently, emerging evidence from in vitro studies and mouse genetics attributed OPG an important role in vascular biology. In fact, OPG could represent the long sought-after molecular link between arterial calcification and bone resorption, which underlies the clinical coincidence of vascular disease and osteoporosis, which are most prevalent in postmenopausal women and elderly people.

Topics: Animals; Arteries; Bone and Bones; Bone Remodeling; Bone Resorption; Calcinosis; Carrier Proteins; Glycoproteins; Humans; Membrane Glycoproteins; Mice; Osteopetrosis; Osteoporosis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Vascular Diseases

Osteoporosis, a disease endemic in Western society, typically reflects an imbalance in skeletal turnover so that bone resorption exceeds bone formation. Bone resorption is the unique function of the osteoclast, and anti-osteoporosis therapy to date has targeted this cell. The osteoclast is a specialized macrophage polykaryon whose differentiation is principally regulated by macrophage colony-stimulating factor, RANK ligand, and osteoprotegerin. Reflecting integrin-mediated signals, the osteoclast develops a specialized cytoskeleton that permits it to establish an isolated microenvironment between itself and bone, wherein matrix degradation occurs by a process involving proton transport. Osteopetrotic mutants have provided a wealth of information about the genes that regulate the differentiation of osteoclasts and their capacity to resorb bone.

Topics: Animals; Bone Resorption; Carrier Proteins; Cell Differentiation; Cell Membrane; Glycoproteins; Humans; Integrins; Macrophage Colony-Stimulating Factor; Macrophages; Membrane Glycoproteins; Osteoclasts; Osteopetrosis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Stromal Cells

Together, osteoporosis and osteopetrosis comprise a substantial proportion of the bone diseases that severely affect humans. In order to understand and effectively treat these disorders, an understanding of the mechanisms controlling bone remodelling is essential. While numerous animal models of bone disease have been generated, the lack of correlation between these animal models and human disease has limited their utility in terms of defining therapeutic strategies. The generation and analysis of cathepsin K knockout mice has resulted in a model for pycnodysostosis, a rare human osteopetrotic disease, and is now providing considerable insights into both osteoclast function and potential therapeutic strategies for the treatment of bone disease. This review highlights the importance of genes such as cathepsin K in understanding bone remodelling and illustrates a new trend towards understanding bone disease as a complete entity rather than as a series of unrelated disorders.

Topics: Animals; Bone Remodeling; Cathepsin K; Cathepsins; Disease Models, Animal; DNA-Binding Proteins; Down Syndrome; Glycoproteins; Humans; Mice; Mice, Knockout; Mutation; Osteopetrosis; Osteoporosis; Osteoprotegerin; Proto-Oncogene Protein c-ets-2; Proto-Oncogene Proteins; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Repressor Proteins; Trans-Activators; Transcription Factors

Topics: Animals; Bone Remodeling; Carrier Proteins; Cytokines; Gene Expression Regulation, Developmental; Glycoproteins; Humans; Ligands; Membrane Glycoproteins; Mice; Mice, Knockout; Mice, Transgenic; Osteoclasts; Osteopetrosis; Osteoporosis; Osteoprotegerin; RANK Ligand; Rats; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Two physiological regulators of osteoclast maturation have recently been identified: the secreted protein osteoprotegerin and the cell-surface ligand to which it binds. These proteins are likely to play an important part in the control of bone resorption, but are also likely to have important roles in other tissues.

Topics: Animals; Antigens, CD; Apoptosis Regulatory Proteins; Bone Remodeling; Calcitriol; Carrier Proteins; Cell Differentiation; Cyclic AMP-Dependent Protein Kinases; Cytokine Receptor gp130; Gene Expression Regulation; Glycoproteins; Humans; Macrophage Colony-Stimulating Factor; Membrane Glycoproteins; Mice; Mice, Mutant Strains; NF-kappa B; Osteoblasts; Osteoclasts; Osteopetrosis; Osteoprotegerin; Parathyroid Hormone; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Receptors, Tumor Necrosis Factor; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Receptor activator of nuclear factor-κB ligand (RANKL) inhibitors are being considered for use in children with osteogenesis imperfecta (OI). We sought to assess efficacy of two doses of a RANKL inhibitor, osteoprotegerin-immunoglobulin Fc segment complex (OPG-Fc), in a growing animal model of OI, the col1α2-deficient mouse (oim/oim) and its wild-type controls (+/+).. Treated mice showed runting and radiographic evidence of osteopetrosis with either high- (20 mg/kg twice weekly) or low-dose (1 mg/kg/week) OPG-Fc. Because of this adverse event, OPG-Fc treatment was halted, and the mice were killed or monitored for recovery with monthly radiographs and assessment of serum osteoclast activity (tartrate-resistant acid phosphatase 5b, TRACP-5b) until 25 wk of age.. Twelve weeks of OPG-Fc treatment resulted in radiographic and histologic osteopetrosis with no evidence of bone modeling and negative tartrate-resistant acid phosphatase staining, root dentin abnormalities, and TRACP-5b activity suppression. Signs of recovery appeared 4-8 wk post-treatment.. Both high- and low-dose OPG-Fc treatment resulted in osteopetrotic changes in infant mice, an outcome that was not seen in studies with the RANKL inhibitor RANK-immunoglobulin Fc segment complex (RANK-Fc) or in studies with older animals. Further investigations of RANKL inhibitors are necessary before their consideration for use in children.

Topics: Acid Phosphatase; Age Factors; Animals; Biomarkers; Bone Remodeling; Collagen Type I; Dentin; Disease Models, Animal; Female; Immunoconjugates; Immunoglobulin Fc Fragments; Isoenzymes; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Osteoclasts; Osteogenesis Imperfecta; Osteopetrosis; Osteoprotegerin; Radiography; RANK Ligand; Risk Assessment; Tartrate-Resistant Acid Phosphatase; Time Factors; Tooth Eruption; Weight Gain

Hematopoietic stem cells (HSCs) are maintained in a specific bone marrow (BM) niche in cavities formed by osteoclasts. Osteoclast-deficient mice are osteopetrotic and exhibit closed BM cavities. Osteoclast activity is inversely correlated with hematopoietic activity; however, how osteoclasts and the BM cavity potentially regulate hematopoiesis is not well understood. To investigate this question, we evaluated hematopoietic activity in three osteopetrotic mouse models: op/op, c-Fos-deficient, and RANKL (receptor activator of nuclear factor kappa B ligand)-deficient mice. We show that, although osteoclasts and, by consequence, BM cavities are absent in these animals, hematopoietic stem and progenitor cell (HSPC) mobilization after granulocyte colony-stimulating factor injection was comparable or even higher in all three lines compared with wild-type mice. In contrast, osteoprotegerin-deficient mice, which have increased numbers of osteoclasts, showed reduced HSPC mobilization. BM-deficient patients and mice reportedly maintain hematopoiesis in extramedullary spaces, such as spleen; however, splenectomized op/op mice did not show reduced HSPC mobilization. Interestingly, we detected an HSC population in osteopetrotic bone of op/op mice, and pharmacological ablation of osteoclasts in wild-type mice did not inhibit, and even increased, HSPC mobilization. These results suggest that osteoclasts are dispensable for HSC mobilization and may function as negative regulators in the hematopoietic system.

Topics: Alendronate; Animals; Bone Density Conservation Agents; Bone Marrow; Cells, Cultured; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cells; Mice; Mice, Transgenic; Osteoclasts; Osteopetrosis; Osteoprotegerin; Proto-Oncogene Proteins c-fos; RANK Ligand; Stem Cell Niche

The receptor activator of nuclear factor-κB (RANK) and its ligand RANKL, which belong to the tumor necrosis factor (TNF) receptor-ligand family, mediate osteoclastogenesis. The crystal structure of the RANKL ectodomain (eRANKL) in complex with the RANK ectodomain (eRANK) combined with biochemical assays of RANK mutants indicated that three RANK loops (Loop1, Loop2, and Loop3) bind to the interface of a trimeric eRANKL. Loop3 is particularly notable in that it is structurally distinctive from other TNF-family receptors and forms extensive contacts with RANKL. The disulfide bond (C125-C127) at the tip of Loop3 is important for determining the unique topology of Loop3, and docking E126 close to RANKL, which was supported by the inability of C127A or E126A mutants of RANK to bind to RANKL. Inhibitory activity of RANK mutants, which contain loops of osteoprotegerin (OPG), a soluble decoy receptor to RANKL, confirmed that OPG shares the similar binding mode with RANK and OPG. Loop3 plays a key role in RANKL binding. Peptide inhibitors designed to mimic Loop3 blocked the RANKL-induced differentiation of osteoclast precursors, suggesting that they could be developed as therapeutic agents for the treatment of osteoporosis and bone-related diseases. Furthermore, some of the RANK mutations associated with autosomal recessive osteopetrosis (ARO) resulted in reduced RANKL-binding activity and failure to induce osteoclastogenesis. These results, together with structural interpretation of eRANK-eRANKL interaction, provided molecular understanding for pathogenesis of ARO.

Topics: Animals; Bone and Bones; Cell Differentiation; Crystallography; Mice; Models, Molecular; Mutagenesis, Site-Directed; Oligopeptides; Osteoclasts; Osteopetrosis; Osteoprotegerin; Peptides, Cyclic; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B

RANKL is an essential mediator of bone resorption, and its activity is inhibited by osteoprotegerin (OPG). Transgenic (Tg) rats were engineered to continuously overexpress OPG to study the effects of continuous long-term RANKL inhibition on bone volume, density, and strength. Lumbar vertebrae, femurs, and blood were obtained from 1-yr-old female OPG-Tg rats (n = 32) and from age-matched wildtype (WT) controls (n = 23). OPG-Tg rats had significantly greater serum OPG (up to 260-fold) and significantly lower serum TRACP5b and osteocalcin compared with WT controls. Vertebral histomorphometry showed significant reductions in osteoclasts and bone turnover parameters in OPG-Tg rats versus WT controls, and these reductions were associated with significantly greater peak load in vertebrae tested through compression. No apparent differences in bone material properties were observed in OPG-Tg rat vertebrae, based on their unchanged intrinsic strength parameters and their normal linear relationship between vertebral bone mass and strength. Femurs from OPG-Tg rats were of normal length but showed mild osteopetrotic changes, including reduced periosteal perimeter (-6%) and an associated reduction in bending strength. Serum OPG levels in WT rats showed no correlations with any measured parameter of bone turnover, mass, or strength, whereas the supraphysiological serum OPG levels in OPG-Tg rats correlated negatively with bone turnover parameters and positively with vertebral bone mass and strength parameters. In summary, low bone turnover after 1 yr of OPG overexpression in rats was associated with increased vertebral bone mass and proportional increases in bone strength, with no evidence for deleterious effects on vertebral material properties.

Topics: Animals; Bone Density; Bone Remodeling; Female; Gene Expression; Lumbar Vertebrae; Organ Size; Osteoclasts; Osteopetrosis; Osteoprotegerin; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Time Factors

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

Topics: Animals; Bone and Bones; Bone Remodeling; Bone Resorption; Cell Differentiation; Fracture Healing; Fractures, Stress; Humans; Osteogenesis; Osteopetrosis; Osteoprotegerin; RANK Ligand; Signal Transduction; Up-Regulation

Autosomal-Recessive Osteopetrosis (ARO) comprises a heterogeneous group of bone diseases for which mutations in five genes are known as causative. Most ARO are classified as osteoclast-rich, but recently a subset of osteoclast-poor ARO has been recognized as due to a defect in TNFSF11 (also called RANKL or TRANCE, coding for the RANKL protein), a master gene driving osteoclast differentiation along the RANKL-RANK axis. RANKL and RANK (coded for by the TNFRSF11A gene) also play a role in the immune system, which raises the possibility that defects in this pathway might cause osteopetrosis with immunodeficiency. From a large series of ARO patients we selected a Turkish consanguineous family with two siblings affected by ARO and hypogammaglobulinemia with no defects in known osteopetrosis genes. Sequencing of genes involved in the RANKL downstream pathway identified a homozygous mutation in the TNFRSF11A gene in both siblings. Their monocytes failed to differentiate in vitro into osteoclasts upon exposure to M-CSF and RANKL, in keeping with an osteoclast-intrinsic defect. Immunological analysis showed that their hypogammaglobulinemia was associated with impairment in immunoglobulin-secreting B cells. Investigation of other patients revealed a defect in both TNFRSF11A alleles in six additional, unrelated families. Our results indicate that TNFRSF11A mutations can cause a clinical condition in which severe ARO is associated with an immunoglobulin-production defect.

Topics: Acid Phosphatase; Actins; Agammaglobulinemia; Amino Acid Sequence; Amino Acid Substitution; Argentina; Arginine; Biopsy; Case-Control Studies; Cell Line, Transformed; Cell Proliferation; Cell Transformation, Viral; Cells, Cultured; Cohort Studies; Consanguinity; Cysteine; Dendrites; DNA Mutational Analysis; Female; Genes, Recessive; Herpesvirus 4, Human; Heterozygote; Homozygote; Humans; Ilium; Isoenzymes; Leukocyte Common Antigens; Leukocytes, Mononuclear; Lipopolysaccharides; Macrophage Colony-Stimulating Factor; Male; Models, Immunological; Molecular Sequence Data; Mutation, Missense; Osteoclasts; Osteopetrosis; Osteoprotegerin; Pakistan; Pedigree; Polymorphism, Genetic; Protein Structure, Tertiary; Radiography, Thoracic; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Vitronectin; Sequence Homology, Amino Acid; Tartrate-Resistant Acid Phosphatase; Turkey

Osteoclasts differentiate from hematopoietic precursors under systemic and local controls. Chemokines and receptors direct leukocyte traffic throughout the body and may help regulate site-specific bone resorption. We investigated bone gene expression in vivo during rapid osteoclast differentiation induced by colony-stimulating factor 1 (CSF-1) in Csf1-null toothless (tl/tl) rats. Long-bone RNA from CSF-1-treated tl/tl rats was analyzed by high-density microarray over a time course. TRAP (tartrate-resistant acid phosphatase)-positive osteoclasts appeared on day 2, peaked on day 4, and decreased slightly on day 6, as marrow space was expanding. TRAP and cathepsin K mRNA paralleled the cell counts. We examined all chemokine and receptor mRNAs on the arrays. CCL9 was strongly induced and peaked on day 2, as did its receptor, CCR1, and regulatory receptors c-Fms (CSF-1 receptor) and RANK (receptor activator of nuclear factor kappaB). Other chemokines and receptors showed little or no significant changes. In situ hybridization and immunohistochemistry revealed CCL9 in small, immature osteoclasts on day 2 and in mature cells at later times. Anti-CCL9 antibody inhibited osteoclast differentiation in culture and significantly suppressed the osteoclast response in CSF-1-treated tl/tl rats. While various chemokines have been implicated in osteoclastogenesis in vitro, this first systematic analysis of chemokines and receptors during osteoclast differentiation in vivo highlights the key role of CCL9 in this process.

Topics: Animals; Cathepsin K; Cathepsins; Cell Differentiation; Cell Proliferation; Chemokines, CC; Gene Expression Profiling; Glycoproteins; Macrophage Colony-Stimulating Factor; Macrophage Inflammatory Proteins; Osteoclasts; Osteopetrosis; Osteoprotegerin; Rats; Rats, Inbred Strains; Receptor, Macrophage Colony-Stimulating Factor; Receptors, CCR1; Receptors, Chemokine; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA

Inactivation of beta-catenin in mesenchymal progenitors prevents osteoblast differentiation; inactivation of Lrp5, a gene encoding a likely Wnt coreceptor, results in low bone mass (osteopenia) by decreasing bone formation. These observations indicate that Wnt signaling controls osteoblast differentiation and suggest that it may regulate bone formation in differentiated osteoblasts. Here, we study later events and find that stabilization of beta-catenin in differentiated osteoblasts results in high bone mass, while its deletion from differentiated osteoblasts leads to osteopenia. Surprisingly, histological analysis showed that these mutations primarily affect bone resorption rather than bone formation. Cellular and molecular studies showed that beta-catenin together with TCF proteins regulates osteoblast expression of Osteoprotegerin, a major inhibitor of osteoclast differentiation. These findings demonstrate that beta-catenin, and presumably Wnt signaling, promote the ability of differentiated osteoblasts to inhibit osteoclast differentiation; thus, they broaden our knowledge of the functions Wnt proteins have at various stages of skeletogenesis.

Topics: Animals; beta Catenin; Bone Development; Cell Differentiation; Cytoskeletal Proteins; Gene Expression Regulation, Developmental; Glycoproteins; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Lac Operon; LDL-Receptor Related Proteins; Low Density Lipoprotein Receptor-Related Protein-5; Mice; Mice, Knockout; Mice, Mutant Strains; Mice, Transgenic; Osteoblasts; Osteoclasts; Osteogenesis; Osteopetrosis; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, LDL; Receptors, Tumor Necrosis Factor; Signal Transduction; Trans-Activators; Wnt Proteins

To investigate the serum creatine kinase isoenzyme pattern, specific biochemical markers of bone metabolism, and cytokines in a Chinese family with osteopetrosis, and correlate abnormalities with the pathophysiology of this condition.. A Chinese female baby was diagnosed with malignant infantile osteopetrosis at the age of 3 weeks by clinical history and biochemical investigations. We studied the laboratory and radiological manifestations of this index case and her family members.. Serum CK-BB fraction of our index patient was elevated to 18.0% (normal 1.6-7.6%). Her biochemical markers of bone resorption including serum C-terminal telopeptide concentration and urine N-terminal telopeptide to creatinine ratio were decreased to 0.54 microg/L (normal 0.72-1.56 microg/L) and 159 x 10(-6) (normal 372-900 x 10(-6)), respectively. Serum cytokines including soluble receptor activator of nuclear factor kappa-B ligand (sRANKL) concentration was suppressed to 0.11 pmol/L (normal 0.23-0.82 pmol/L) and osteoprotegerin (OPG) concentration was 4.9 pmol/L (normal 2.8-4.9 pmol/L), resulting in an elevated OPG to sRANKL ratio of 44.5 (normal 3.8-19.4) in favour of bone formation.. If left untreated, this condition is usually fatal within the first year of life. With early diagnosis, management including bone marrow transplantation can be planned ahead and will result in a better survival.

Topics: Adult; Biomarkers; Bone and Bones; Bone Marrow Transplantation; Carrier Proteins; China; Creatine Kinase; Cytokines; Female; Fetal Blood; Glycoproteins; Humans; Infant, Newborn; Isoenzymes; Male; Membrane Glycoproteins; Osteopetrosis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Topics: Animals; Bone Density; Bone Remodeling; Calcification, Physiologic; Disease Models, Animal; Glycoproteins; Macrophage Colony-Stimulating Factor; Mice; Mice, Knockout; Osteopetrosis; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tibia

We report on a case of osteoclast-poor osteopetrosis who received a hematopoietic stem cell graft and, despite hematological engraftment, showed little signs of response in the skeletal defect. Clinical and laboratory studies supported the concept that the bone microenvironment remained abnormal, thus reducing the clinical response to transplantation.. Osteopetrosis is a rare genetic disorder characterized by severely reduced bone resorption resulting from a defect in either osteoclast development (osteoclast-poor osteopetrosis) or activation (osteoclast-rich osteopetrosis). Patients with osteoclast-rich osteopetrosis can be rescued by allogenic hematopoietic stem cell transplantation; however, little information exists concerning the success of transplantation as a treatment for osteoclast-poor osteopetrosis. We report on a child with osteoclast-poor osteopetrosis whose diagnosis was delayed, consequently receiving a cord blood transplant from an unrelated donor at the age of 8 years. Engraftment was deemed successful by peripheral blood genotyping, although >3 years after transplantation there was little rescue of the skeletal defect and anemia, and extramedullary hematopoiesis persisted.. Peripheral blood mononuclear cells from the osteopetrosis patient, before and after transplantation, were used to generate osteoclasts in vitro in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL.. Before transplantation few, small mononuclear osteoclasts formed (F-actin ring-positive cells, co-localizing with vitronectin receptor [alphavbeta3 integrin] and TRACP) associated with occasional, small resorption lacunae. Low levels of collagen C-terminal telopeptide (CTx) fragments were released from these cultures as assessed by ELISA (CrossLaps; patient, 12.85 nM; control, 448.6 nM). In contrast, osteoclasts formed in cultures after transplantation formed to a similar degree to control cultures from healthy individuals: large numbers of osteoclasts containing numerous nuclei were present, and approximately 50% of the surface of bone slices was resorbed, associated with intermediate levels of collagen fragment release (116.48 nM). The culture data reflect the histopathology and radiological findings and also support previous studies showing that neither M-CSF nor RANKL rescues osteoclast-poor osteopetrosis.. This is the first case reported in which a successful hematopoietic engraftment failed to correct an osteopetrotic skeletal defect, and this finding may be credited to the age at which the child was transplanted.

Topics: Acid Phosphatase; Biopsy; Carrier Proteins; Cartilage; CD11c Antigen; CD18 Antigens; Cell Differentiation; Child; Collagen; Collagen Type I; Cord Blood Stem Cell Transplantation; DNA Mutational Analysis; Female; Femur; Glycoproteins; Hematologic Diseases; Humans; Humerus; Integrin alphaVbeta3; Isoenzymes; Leukocytes, Mononuclear; Macrophage Colony-Stimulating Factor; Membrane Glycoproteins; Osteoclasts; Osteopetrosis; Osteoprotegerin; Peptides; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tartrate-Resistant Acid Phosphatase; Transplantation, Homologous; Treatment Outcome

Osteoclasts are derived from hemopoietic stem cells and play critical roles in bone resorption and remodeling. Multinucleated osteoclasts are attached tightly to bone matrix, whereas precursor cells with the potential to differentiate into osteoclasts in culture are widely distributed. In this study, we assessed the characteristics of osteoclast precursors in bone marrow (BM) and in extramedullary organs as indicated by their responsiveness to ligands for Toll-like receptors (TLRs) and to TNF-alpha. Development of osteoclasts from precursor cells in the BM was inhibited by CpG oligonucleotides, a ligand for TLR9, but not by LPS, a ligand for TLR4. BM osteoclasts were induced by TNF-alpha as well as receptor activator of NF-kappaB ligand in the presence of M-CSF. Splenic osteoclast precursors, even in osteoclast-deficient osteopetrotic mice, differentiated into mature osteoclasts following exposure to TNF-alpha or receptor activator of NF-kappaB ligand. However, splenic osteoclastogenesis was inhibited by both LPS and CpG. Osteoclastogenesis from peritoneal precursors was inhibited by not only these TLR ligands but also TNF-alpha. The effects of peptidoglycan, a ligand for TLR2, were similar to those of LPS. BM cells precultured with M-CSF were characterized with intermediate characteristics between those of splenic and peritoneal cavity precursors. Taken together, these findings demonstrate that osteoclast precursors are not identical in the tissues examined. To address the question of why mature osteoclasts occur only in association with bone, we may characterize not only the microenvironment for osteoclastogenesis, but also the osteoclast precursor itself in intramedullary and extramedullary tissues.

Topics: Animals; Ascitic Fluid; Bone Marrow Cells; Carrier Proteins; Cell Differentiation; Cell Line; Cells, Cultured; Clone Cells; Glycoproteins; Growth Inhibitors; Immune Tolerance; Injections, Intravenous; Ligands; Lipopolysaccharides; Macrophage Colony-Stimulating Factor; Macrophages; Membrane Glycoproteins; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Transgenic; Oligodeoxyribonucleotides; Osteoclasts; Osteopetrosis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Signal Transduction; Spleen; Stem Cells; Stromal Cells; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors; Tumor Necrosis Factor-alpha

Klotho gene mutant mice (klotho mice, also called kl/kl) exhibit osteopetrosis in the metaphysis of femora and tibiae and die within 3 months. We previously showed by semiquantitative RT-PCR that osteoprotegerin (opg) expression levels in klotho mice were about 2-fold higher than those in wild-type mice in the bone marrow, spleen, and lung. To examine whether the high osteoprotegerin expression levels account for the osteopetrotic phenotype in the klotho homozygous mutant mice in vivo, we made double mutant mice by crossing klotho mutant and osteoprotegerin-deficient mice. Micro computed tomography analysis in the two-dimensional sagittal planes of the metaphyses and cross-sections of femoral midshaft revealed that the abnormally high fractional trabecular bone volume in klotho homozygous mice (kl/kl; 29.71%), which was about 4-fold higher compared with that of wild-type [klotho (+/+) opg (+/+)] mice (7.81%), was rescued by the coexistence of heterozygous mutation in opg gene locus (+/-; 8.36%). Single heterozygous mutation in the opg gene locus alone (without klotho mutation) did not show phenotype (trabecular bone volume, 5.84%; not significantly different from wild type). High levels of osteoprotegerin mRNA expression in the bone marrow in klotho mutant mice were reduced by the heterozygous mutation in the opg gene locus. Furthermore, high osteoprotegerin protein levels in klotho mutant mice were also reduced by the heterozygous mutations in opg gene locus. Thus, elevated levels of osteoprotegerin in mutant mice contribute at least in part to reveal the osteopetrotic phenotype in klotho mice.

Topics: Animals; B-Lymphocytes; Bone Marrow Cells; Enzyme-Linked Immunosorbent Assay; Femur; Flow Cytometry; Glucuronidase; Glycoproteins; Hematopoiesis; Heterozygote; Klotho Proteins; Lymphopenia; Membrane Proteins; Mice; Mice, Knockout; Mutation; Osteopetrosis; Osteoprotegerin; Phenotype; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spleen; Tomography, X-Ray Computed

High systemic levels of osteoprotegerin (OPG) in OPG transgenic mice cause osteopetrosis with normal tooth eruption and bone elongation and inhibit the development and activity of endosteal, but not periosteal, osteoclasts. We demonstrate that both intravenous injection of recombinant OPG protein and transgenic overexpression of OPG in OPG(-/-) mice effectively rescue the osteoporotic bone phenotype observed in OPG-deficient mice. However, intravenous injection of recombinant OPG over a 4-wk period could not reverse the arterial calcification observed in OPG(-/-) mice. In contrast, transgenic OPG delivered from mid-gestation through adulthood does prevent the formation of arterial calcification in OPG(-/-) mice. Although OPG is normally expressed in arteries, OPG ligand (OPGL) and receptor activator of NF-kappaB (RANK) are not detected in the arterial walls of wild-type adult mice. Interestingly, OPGL and RANK transcripts are detected in the calcified arteries of OPG(-/-) mice. Furthermore, RANK transcript expression coincides with the presence of multinuclear osteoclast-like cells. These findings indicate that the OPG/OPGL/RANK signaling pathway may play an important role in both pathological and physiological calcification processes. Such findings may also explain the observed high clinical incidence of vascular calcification in the osteoporotic patient population.

Topics: Acid Phosphatase; Animals; Aorta; Blotting, Western; Bone Density; Calcinosis; Cathepsin K; Cathepsins; CHO Cells; Cricetinae; Femur; Glycoproteins; Humans; Immunohistochemistry; In Situ Hybridization; Isoenzymes; Mice; Mice, Knockout; Mice, Transgenic; NF-kappa B; Osteoclasts; Osteopetrosis; Osteoporosis; Osteoprotegerin; Radiography; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Fusion Proteins; Tartrate-Resistant Acid Phosphatase

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

A novel secreted glycoprotein that regulates bone resorption has been identified. The protein, termed Osteoprotegerin (OPG), is a novel member of the TNF receptor superfamily. In vivo, hepatic expression of OPG in transgenic mice results in a profound yet nonlethal osteopetrosis, coincident with a decrease in later stages of osteoclast differentiation. These same effects are observed upon administration of recombinant OPG into normal mice. In vitro, osteoclast differentiation from precursor cells is blocked in a dose-dependent manner by recombinant OPG. Furthermore, OPG blocks ovariectomy-associated bone loss in rats. These data show that OPG can act as a soluble factor in the regulation of bone mass and imply a utility for OPG in the treatment of osteoporosis associated with increased osteoclast activity.

Topics: Amino Acid Sequence; Animals; Bone Density; Bone Resorption; Cell Differentiation; Cells, Cultured; Cricetinae; Female; Gene Expression Regulation, Developmental; Glycoproteins; Humans; Liver; Male; Mice; Mice, Transgenic; Molecular Sequence Data; Organ Specificity; Osteoclasts; Osteopetrosis; Osteoprotegerin; Ovariectomy; Rats; Rats, Inbred F344; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Fusion Proteins; RNA, Messenger; Sequence Homology, Amino Acid