osteoprotegerin and Hypercalcemia

Topics: Animals; Antibodies, Monoclonal, Humanized; Bone and Bones; Bone Neoplasms; Bone Resorption; Denosumab; Disease Models, Animal; Drug Design; Humans; Hypercalcemia; Mice; Molecular Targeted Therapy; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B

Topics: Animals; Bone Density Conservation Agents; Calcitonin; Diphosphonates; Fluid Therapy; Humans; Hypercalcemia; Neoplasms; Osteoprotegerin; Paraneoplastic Syndromes; Parathyroid Hormone-Related Protein; Prednisolone; Sodium; Sodium Potassium Chloride Symporter Inhibitors

Hypercalcemia arises in the advanced stage of myeloma due to marked increase in osteoclastic bone resorption by local factors in the bone marrow, and is among the most serious complications in myeloma. It can progress rapidly and provoke acute renal failure and coma, which may become life-threatening. Early detection by serum calcium levels and treatment of hypercalcemia is important, although its clinical symptoms are difficult to distinguish from those in other underlying complications. Bisphosphonates, potent inhibitors of bone resorption, are recommended as the most effective therapy and have been approved for treatment of hypercalcemia in myeloma.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Biomarkers; Bone Density Conservation Agents; Bone Resorption; Calcium; Denosumab; Diphosphonates; Humans; Hypercalcemia; Multiple Myeloma; Osteoclasts; Osteoprotegerin; RANK Ligand

Malignancy-associated hypercalcemia (MAH) is caused by tumor over-production of parathyroid hormone-related protein (PTHrP), or by locally enhanced bone resorption in metastatic lesions of solid cancers. Medical treatment of MAH includes hydration by saline infusion, loop diuretics to promote urinary calcium excretion and anti-resorptives such as calcitonin and bisphosphonates. Particularly, bisphosphonates are the current mainstay for MAH treatment : they not only inhibit osteoclastic bone resorption to ameliorate hypercalcemia but can also alleviate bone pain and can even prevent cancer cell expansion in bone metastatic lesions.

Topics: Bone Neoplasms; Bone Resorption; Calcitonin; Diphosphonates; Drug Design; Drug Therapy, Combination; Fluid Therapy; Glycoproteins; Humans; Hypercalcemia; Neoplasms; Osteoprotegerin; Parathyroid Hormone-Related Protein; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Sodium Chloride; Sodium Potassium Chloride Symporter Inhibitors

Topics: Animals; Antibodies; Cholecalciferol; Diagnosis, Differential; Humans; Hypercalcemia; Osteoprotegerin; Palliative Care; Paraneoplastic Endocrine Syndromes; Parathyroid Hormone-Related Protein; Prognosis

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

Topics: Animals; Apoptosis; Arthritis, Experimental; Carrier Proteins; Glycoproteins; Humans; Hypercalcemia; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Knockout; Mice, Transgenic; Neoplasms; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; T-Lymphocytes

The influence of sodium fluoride (NaF) on calcium metabolism was examined in goldfish (fresh water teleost). At 2days after administration of NaF (500ng/g body weight; 5μg/g body weight) (around 10(-5) to 10(-4)M in goldfish), we indicated that plasma calcium levels upregulated in both doses of NaF-treated goldfish. To examine the mechanism of hypercalcemia by NaF treatments, therefore, direct effects of NaF on osteoblasts and osteoclasts in goldfish were investigated by an original assay system using teleost scale which has osteoblasts, osteoclasts and bone matrix. Alkaline phosphatase activity in the scales increased with the treatment of NaF (10(-6) and 10(-5)M) during 6h of incubation. Also, tartrate-resistant acid phosphatase activity increased after exposure to NaF (10(-5)M) at the 6h of incubation. To investigate the osteoclastic activation, the mRNA expression of osteoclastogenesis related factors were examined. The receptor activator of the nuclear factor-κB ligand (RANKL) which is known as a factor for osteoclastogenesis, increased in the NaF-treated scales after 6h of incubation. The ratio of RANKL/osteoprotegerin (osteoclastogenesis inhibitory factor) significantly increased after 6h of incubation. Resulting from the increase of RANKL mRNA level, the expression of transcription-regulating factors was significantly increased. Furthermore, the expression of functional genes, cathepsin K and matrix metalloproteinase-9 mRNA, was significantly increased. In our knowledge, this is the first report concerning the effects of NaF on osteoblasts and osteoclasts in teleosts. We concluded that NaF influences calcium metabolism via osteoclastic activation in goldfish.

Topics: Alkaline Phosphatase; Animals; Calcium; Cathepsin K; Dose-Response Relationship, Drug; Fish Proteins; Goldfish; Hypercalcemia; Matrix Metalloproteinase 9; Osteoblasts; Osteoclasts; Osteoprotegerin; RANK Ligand; RNA, Messenger; Sodium Fluoride; Tartrate-Resistant Acid Phosphatase; Time Factors; Transcription, Genetic; Water Pollutants, Chemical

Hypercalcemia associated with myelofibrosis is rare, and its pathogenesis and treatment are not known.. We report a unique case of hypercalcemia associated with post-essential thrombocythemia myelofibrosis and review the clinical and laboratory features, pathogenesis, and responsiveness to treatment with the bone antiresorptive agent, denosumab.. A 62-yr-old woman with essential thrombocythemia presented with progression to myelofibrosis with lytic skull lesions and symptomatic hypercalcemia. Other causes of hypercalcemia were excluded. Her disturbance in calcium homeostasis was not PTH- or vitamin D-mediated, although this has been postulated in cases of hypercalcemia with the related entity of primary myelofibrosis. Her hypercalcemia was refractory to aggressive iv saline administration, furosemide, calcitonin, and pamidronate, but promptly improved after one 120-mg sc dose of the anti-receptor activator of nuclear factor κB (RANK) ligand monoclonal antibody, denosumab, with sustained normocalcemia for approximately 2 months. She died 6 months later from complications due to the leukemic transformation of her hematological disease.. The pathogenesis of myelofibrosis-related hypercalcemia could be due to multiple factors, particularly changes in the RANK ligand-RANK-osteoprotegerin system that lead to increased osteoclast activity. Although we did not measure these factors, denosumab holds promise in the treatment of malignancy-associated hypercalcemia and specifically that related to myelofibrosis. Hypercalcemia associated with myelofibrosis is rare, and its pathogenesis and treatment are not known.

Topics: Antibodies, Monoclonal, Humanized; Autopsy; Biopsy; Bone Marrow; Calcium; Denosumab; Fatal Outcome; Female; Humans; Hypercalcemia; Leukemia; Middle Aged; Osteoprotegerin; Primary Myelofibrosis; RANK Ligand; Thrombocytosis

p38 mitogen-activated protein kinase (MAPK) acts downstream in the signaling pathway that includes receptor activator of NF-κB (RANK), a powerful inducer of osteoclast formation and activation. We investigated the role of p38 MAPK in parathyroid hormone related protein (PTHrP)-induced osteoclastogenesis in vitro and PTHrP-induced bone resorption in vivo. The ability of FR167653 to inhibit osteoclast formation was evaluated by counting the number of tartrate-resistant acid phosphatase positive multinucleated cells (TRAP-positive MNCs) in in vitro osteoclastgenesis assays. Its mechanisms were evaluated by detecting the expression level of c-Fos and nuclear factor of activated T cells c1 (NFATc1) in bone marrow macrophages (BMMs) stimulated with sRANKL and M-CSF, and by detecting the expression level of osteoprotegerin (OPG) and RANKL in bone marrow stromal cells stimulated with PTHrP in the presence of FR167653. The function of FR167653 on bone resorption was assessed by measuring the bone resorption area radiographically and by counting osteoclast number per unit bone tissue area in calvaria in a mouse model of bone resorption by injecting PTHrP subcutaneously onto calvaria. Whole blood ionized calcium levels were also recorded. FR167653 inhibited PTHrP-induced osteoclast formation and PTHrP-induced c-Fos and NFATc1 expression in bone marrow macrophages, but not the expression levels of RANKL and OPG in primary bone marrow stromal cells treated by PTHrP. Furthermore, bone resorption area and osteoclast number in vivo were significantly decreased by the treatment of FR167653. Systemic hypercalcemia was also partially inhibited. Inhibition of p38 MAPK by FR167653 blocks PTHrP-induced osteoclastogenesis in vitro and PTHrP-induced bone resorption in vivo, suggesting that the p38 MAPK signaling pathway plays a fundamental role in PTHrP-induced osteoclastic bone resorption.

Topics: Acid Phosphatase; Animals; Bone Resorption; Calcium; Cell Count; Cells, Cultured; Humans; Hypercalcemia; Isoenzymes; Male; Mice; NFATC Transcription Factors; Osteoclasts; Osteogenesis; Osteoprotegerin; p38 Mitogen-Activated Protein Kinases; Parathyroid Hormone-Related Protein; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-fos; Pyrazoles; Pyridines; RANK Ligand; Tartrate-Resistant Acid Phosphatase

RANKL is a TNF family member that mediates osteoclast formation, activation, and survival by activating RANK. The proresorptive effects of RANKL are prevented by binding to its soluble inhibitor osteoprotegerin (OPG). Recombinant human OPG-Fc recognizes RANKL from multiple species and reduced bone resorption and increased bone volume, density, and strength in a number of rodent models of bone disease. The clinical development of OPG-Fc was discontinued in favor of denosumab, a fully human monoclonal antibody that specifically inhibits primate RANKL. Direct binding assays showed that denosumab bound to human RANKL but not to murine RANKL, human TRAIL, or other human TNF family members. Denosumab did not suppress bone resorption in normal mice or rats but did prevent the resorptive response in mice challenged with a human RANKL fragment encoded primarily by the fifth exon of the RANKL gene. To create mice that were responsive to denosumab, knock-in technology was used to replace exon 5 from murine RANKL with its human ortholog. The resulting "huRANKL" mice exclusively express chimeric (human/murine) RANKL that was measurable with a human RANKL assay and that maintained bone resorption at slightly reduced levels versus wildtype controls. In young huRANKL mice, denosumab and OPG-Fc each reduced trabecular osteoclast surfaces by 95% and increased bone density and volume. In adult huRANKL mice, denosumab reduced bone resorption, increased cortical and cancellous bone mass, and improved trabecular microarchitecture. These huRANKL mice have potential utility for characterizing the activity of denosumab in a variety of murine bone disease models.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibody Affinity; Antibody Specificity; Bone and Bones; Bone Density; Bone Resorption; Denosumab; Gene Knock-In Techniques; Humans; Hypercalcemia; Mice; Molecular Sequence Data; Osteoclasts; Osteogenesis; Osteoprotegerin; Phenotype; Protein Binding; RANK Ligand; X-Ray Microtomography

Human T-cell leukemia virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATL), frequently associated with hypercalcemia and bone destruction. A positive correlation between the appearance of an antibody recognizing the central region (Asp197 to Leu216) on Gp46, gp46-197, and the severity of ATL has been demonstrated. In this study, five male Nihon Hakusyoku rabbits were immunized with a synthetic peptide corresponding to the gp46-197 region to clarify its action and mechanism. Two of the rabbits showed piloerection, anorexia, and somnolence, and died soon after booster administration. The serum calcium level of the dead rabbits was significantly high, compared to those of surviving rabbits. Interestingly, amino acid sequences homologous with gp46-197 were found in the carboxyl-terminal half of osteoprotegerin (OPG), an osteoclast inhibitory factor. To confirm the effect of the gp46-197 region on osteogenesis in vivo, the peptide was intraperitoneally administered to male Sprague-Dawley rats. The administration of the gp46-197 peptide resulted in a decrease of bone mineral density (BMD), a significant increase of serum calcium level, and inhibition of normal bone growth in both short- and long-term experiments. In rats, femoral growth inhibition by the gp46-197 peptide was restored by the coadministration of recombinant human OPG. Improvement by OPG in the adverse effect indicates that the central region of HTLV-1 Gp46 acts as an antagonist for OPG and leads to hypercalcemia.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Chromatography, High Pressure Liquid; Enzyme-Linked Immunosorbent Assay; Gene Products, env; HTLV-I Antibodies; Humans; Hypercalcemia; Leukemia-Lymphoma, Adult T-Cell; Male; Molecular Mimicry; Molecular Sequence Data; Osteoprotegerin; Rabbits; Rats; Rats, Sprague-Dawley; Retroviridae Proteins, Oncogenic; Sequence Homology, Amino Acid

2-Methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D3 (2MD), an analog of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], has been shown to strongly induce bone formation both in vitro and in vivo. We have synthesized four substituents at carbon 2 of 2MD (2MD analogs), four stereoisomers at carbon 20 of the respective 2MD analogs (2MD analog-C20 isomers) and four 2MD analogs with an oxygen atom at carbon 22 (2MD-22-oxa analogs) and examined their ability to stimulate osteoclastogenesis and induce hypercalcemia. 2MD analogs were 100 times as potent as 1alpha,25(OH)2D3 in stimulating the formation of osteoclasts in vitro and in inducing the expression of receptor activator of NF-kappaB ligand (RANKL) and 25-hydroxyvitamin D3-24 hydroxylase mRNAs in osteoblasts. The osteoclast-inducing activities of 2MD analog-C20 isomers and 2MD 22-oxa analogs were much weaker than those of 2MD analogs. In addition, the activity of a 2MD analog in inducing dentine resorption was much stronger than that of 1alpha,25(OH)2D3 in the pit formation assay. Affinities to the vitamin D receptor and transcriptional activities of these compounds did not always correlate with their osteoclastogenic activities. Osteoprotegerin-deficient (OPG-/-) mice provide a suitable model for investigating in vivo effects of 2MD analogs because they exhibit extremely high concentrations of serum RANKL. The same amounts of 2MD analogs and 1alpha,25(OH)2D3 were administered daily to OPG-/- mice for 2 days. The elevation in serum concentrations of RANKL and calcium was much greater in 2MD analog-treated OPG-/- mice than in 1alpha,25(OH)2D3-treated ones. A 2MD analog was much more potent than 1alpha,25(OH)2D3 in causing hypercalcemia and in increasing soluble RANKL with enhanced osteoclastogenesis even in wild-type mice. In contrast, the administration of the 2MD analog to c-fos-deficient mice failed to induce osteoclastogenesis and hypercalcemia. These results suggest that new substituents at carbon 2 of 2MD strongly stimulate osteoclast formation in vitro and in vivo, and that osteoclastic bone resorption is indispensable for their hypercalcemic action of 2MD analogs in vivo.

Topics: Animals; Animals, Newborn; Calcitriol; Cells, Cultured; Hypercalcemia; Male; Mice; Mice, Knockout; Osteoblasts; Osteoclasts; Osteoprotegerin; Proto-Oncogene Proteins c-fos; RANK Ligand; Receptors, Calcitriol; RNA, Messenger; Steroid Hydroxylases; Structure-Activity Relationship; Vitamin D3 24-Hydroxylase

Receptor activator of NF-kappaB (RANK) and its ligand (RANKL) are essential for osteoclast formation, function, and survival. Osteoprotegerin (OPG) inhibits RANK signaling by sequestering RANKL. This study evaluated the antiosteoclast and immunoregulatory effects of mouse rRANK-Fc, which, similar to OPG, can bind RANKL. The effect of RANKL inhibition by RANK-Fc on osteoclast function was determined by inhibition of vitamin D(3) (1,25(OH)(2)D(3))-induced hypercalcemia. Mice were injected with a single dose of 0, 10, 100, 500, or 1000 microg of RANK-Fc; 100 microg of OPG-Fc; or 5 microg of zoledronate 2 h before 1,25(OH)(2)D(3) challenge on day 0, and sacrificed on days 1, 2, 4, 6, 8, 12, 16, and 20. RANK-Fc doses of 100 or 500 microg were tested in a mouse respiratory influenza virus host-resistance model. A single dose of RANK-Fc > or =100 microg suppressed elevation of serum calcium levels and suppressed the bone turnover marker serum pyridinoline at day 4 and later time points, similar to those observed with OPG-Fc and zoledronate (p < or = 0.01 vs controls). By day 6, both immature and mature osteoclasts were depleted by high doses of RANK-Fc (500 and 1000 microg) or 100 microg of OPG-Fc. RANK-Fc doses of 100 or 500 microg had no detectable effect on immune responses to influenza infection, as measured by activation of cytotoxic T cell activity, influenza-specific IgG response, and virus clearance. RANK-Fc inhibition of RANKL has antiosteoclast activity at doses that have no detectable immunoregulatory activity, suggesting that RANKL inhibitors be further studied for their potential to treat excess bone loss.

Topics: Animals; Bone Resorption; Diphosphonates; Disease Models, Animal; Dose-Response Relationship, Immunologic; Female; Hydroxycholecalciferols; Hypercalcemia; Imidazoles; Immunity, Innate; Immunoglobulin G; Influenza A Virus, H3N2 Subtype; Longitudinal Studies; Male; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Recombinant Fusion Proteins; Zoledronic Acid

We have previously demonstrated that parathyroid hormone-related protein (PTHrP) is a cachexia inducer, but it is still not known what PTHrP effects on target tissues induce the cachexia. Therefore, we examined the effects of anti-PTHrP antibody and osteoprotegerin (OPG) on PTHrP-producing tumor-induced cachexia. Nude mice bearing PTHrP-producing human lung cancer cells (HARA-B) exhibited cachexia with hypercalcemia 3-4 weeks after inoculation, accompanied by losses in body, adipose tissue, and muscle weight. OPG ameliorated hypercalcemia, as did neutralization of PTHrP with antibody; and it increased both body and adipose tissue weights. These increases in body and adipose tissue weight, however, were significantly less than those in mice treated with anti-PTHrP antibody. Simultaneous administration of OPG and anti-PTHrP antibody caused significant increases in body, adipose tissue, and muscle weight, along with an immediate decrease in blood ionized calcium levels. The increase in body weight was similar to that observed in mice treated with anti-PTHrP antibody alone, and the decrease in the blood ionized calcium levels was significantly greater than that in mice treated with OPG or anti-PTHrP antibody alone. These results suggest that an effect of PTHrP on target tissues other than hypercalcemia is involved in the development of cachexia. Expression of cachexia-inducing proinflammatory cytokines (interleukin-6 and leukemia inhibitory factor) is stimulated by PTHrP. This might be a mechanism by which PTHrP produces tumor-induced cachexia. It is also suggested that OPG and anti-PTHrP antibody synergistically act to ameliorate hypercalcemia, although the mechanism responsible for this is unclear.

Topics: Animals; Antibodies, Monoclonal; Body Weight; Cachexia; Disease Models, Animal; Glycoproteins; Humans; Hypercalcemia; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Osteoprotegerin; Parathyroid Hormone-Related Protein; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tumor Cells, Cultured

Humoral hypercalcemia of malignancy (HHM) is mediated primarily by skeletal and renal responses to tumor-derived PTHrP. PTHrP mobilizes calcium from bone by inducing the expression of receptor activator for nuclear factor-kappaB ligand (RANKL), a protein that is essential for osteoclast formation, activation, and survival. RANKL does not influence renal calcium reabsorption, so RANKL inhibition is a rational approach to selectively block, and thereby reveal, the relative contribution of bone calcium to HHM. We used the RANKL inhibitor osteoprotegerin (OPG) to evaluate the role of osteoclast-mediated hypercalcemia in two murine models of HHM. Hypercalcemia was induced either by sc inoculation of syngeneic colon (C-26) adenocarcinoma cells or by sc injection of high-dose recombinant PTHrP (0.5 mg/kg, s.c., twice per day). In both models, OPG (0.2-5 mg/kg) caused rapid reversal of established hypercalcemia, and the speed and duration of hypercalcemia suppression were significantly greater with OPG (5 mg/kg) than with high-dose bisphosphonates (pamidronate or zoledronic acid, 5 mg/kg). OPG also caused greater reductions in osteoclast surface and biochemical markers of bone resorption compared with either bisphosphonate. In both models, hypercalcemia gradually returned despite clear evidence of ongoing suppression of bone resorption by OPG. These data demonstrate that osteoclasts and RANKL are important mediators of HHM, particularly in the early stages of the condition. Aggressive antiresorptive therapy with a RANKL inhibitor therefore might be a rational approach to controlling HHM.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Bone Resorption; Calcium; Carrier Proteins; Cell Line, Tumor; Colonic Neoplasms; Diphosphonates; Disease Models, Animal; Glycoproteins; Humans; Hypercalcemia; Ligands; Membrane Glycoproteins; Mice; NF-kappa B; Osteoprotegerin; Pamidronate; Parathyroid Hormone-Related Protein; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

IL-4 is an important immune cytokine that regulates bone homeostasis. We investigated the molecular mechanism of IL-4 action on bone-resorbing mature osteoclasts. Using a highly purified population of mature osteoclasts, we show that IL-4 dose-dependently inhibits receptor activator of NF-kappaB ligand (RANKL)-induced bone resorption by mature osteoclasts. We detected the existence of IL-4R mRNA in mature osteoclasts. IL-4 decreases TRAP expression without affecting multinuclearity of osteoclasts, and inhibits actin ring formation and migration of osteoclasts. Interestingly, IL-4 inhibition of bone resorption occurs through prevention of RANKL-induced nuclear translocation of p65 NF-kappaB subunit, and intracellular Ca(2+) changes. Moreover, IL-4 rapidly decreases RANKL-stimulated ionized Ca(2+) levels in the blood, and mature osteoclasts in IL-4 knockout mice are sensitive to RANKL action to induce bone resorption and hypercalcemia. Furthermore, IL-4 inhibits bone resorption and actin ring formation by human mature osteoclasts. Thus, we reveal that IL-4 acts directly on mature osteoclasts and inhibits bone resorption by inhibiting NF-kappaB and Ca(2+) signaling.

Topics: Acid Phosphatase; Actins; Active Transport, Cell Nucleus; Adult; Animals; Bone Resorption; Calcium Signaling; Carrier Proteins; Cell Differentiation; Cell Migration Inhibition; Glycoproteins; Humans; Hypercalcemia; Interleukin-4; Intracellular Fluid; Isoenzymes; Male; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Knockout; NF-kappa B; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Calcitonin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA, Messenger; Tartrate-Resistant Acid Phosphatase; Transcription Factor RelA

Topics: Animals; Carrier Proteins; Cell Death; Glycoproteins; Hypercalcemia; Membrane Glycoproteins; Mice; Models, Biological; Neoplasms; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Insufficient dietary magnesium (Mg) intake has been associated in humans with low bone mass. Mg deficiency in the rat has suggested bone loss is due to increased bone resorption and/or inadequate bone formation during remodeling. The purpose of this study was to assess the effect of a low Mg diet on bone and mineral metabolism in the young and mature BALB/c mouse and explore the hypothesis that inflammatory cytokines may contribute to Mg deficiency-induced osteoporosis. Using an artificial diet, we induced targeted Mg depletion (0.002% Mg) with all other nutrients maintained at the normal level. In all Mg-depleted mice, hypomagnesemia developed and skeletal Mg content fell significantly. The serum Ca in Mg-deficient mice was higher than in control mice; however, serum PTH levels were not significantly different. Osteoprotegerin (OPG) in dosages that inhibit osteoclastic bone resorption did not prevent hypercalcemia in Mg-deficient animals. No significant difference in serum Ca was observed between groups when dietary Ca was reduced by 50%, suggesting that a compensatory increase in intestinal absorption might account for the hypercalcemia. Growth plate width decreased 33% in young Mg-deficient animals and chondrocyte columns decreased in number and length, suggesting that Mg deficiency reduced bone growth. Trabecular bone volume in the metaphysis of the tibia in these animals was decreased and osteoclast number was increased by 135%. Osteoblast number was significantly reduced. Immunohistochemistry revealed that substance P increased 230% and 200% in megakaryocytes and lymphocytes, respectively, after 1 day of Mg depletion. IL-1 increased by 140% in osteoclasts by day 3 and TNF alpha increased in osteoclasts by 120% and 500% in megakaryocytes on day 12. This study demonstrates a profound effect of Mg depletion on bone characterized by impaired bone growth, decreased osteoblast number, increased osteoclast number in young animals, and loss of trabecular bone with stimulation of cytokine activity in bone.

Topics: Animals; Bone Resorption; Calcium; Cytokines; Diet; Disease Models, Animal; Female; Femur; Glycoproteins; Growth Plate; Hypercalcemia; Hypocalcemia; Injections, Subcutaneous; Magnesium; Magnesium Deficiency; Mice; Mice, Inbred BALB C; Minerals; Osteoclasts; Osteoporosis; Osteoprotegerin; Parathyroid Hormone; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tibia

In the present study, we used osteoprotegerin (OPG), which blocks osteoclastogenesis, to correct and thus explain the hypercalcemia that is seen during dietary Mg deficiency in the mouse. Control and Mg-deficient mice received injections for 12 days of either OPG or vehicle only. Serum Ca was similar in Mg-deficient mice treated with OPG and in control mice receiving OPG (9.2 +/- 0.3 mg/dl vs. 9.2 +/- 0.5). Both groups had significantly higher serum Ca than controls or Mg-deficient animals receiving vehicle alone. Surprisingly, Mg-depleted mice that received OPG in doses that inhibit osteoclastic bone resorption remained hypercalcemic. Because mature osteoclasts still present in the marrow might be hyperactive, we examined osteoclast morphology at the light microscopic and ultrastructural level. Light microscopic examination of trabecular bone showed few osteoclasts in OPG-treated mice. Ultrastructural examination revealed that osteoclasts in OPG-treated mice have decreased contact with the endosteal bone surface and absence of a ruffled border. Because the morphology of the existing pool of mature osteoclasts did not enhance resorption, another mechanism, such as increased intestinal absorption of Ca in Mg-deficient mice, likely contributes to the hypercalcemia observed during Mg deficiency.

Topics: Acid Phosphatase; Animals; Calcium; Female; Glycoproteins; Hypercalcemia; Isoenzymes; Magnesium Deficiency; Mice; Osteoclasts; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tartrate-Resistant Acid Phosphatase

Hypercalcemia is one of the most frequent and serious complications in patients with adult T-cell leukemia (ATL) and is due to marked bone resorption by accumulation of osteoclasts (OCLs). Although several cytokines such as interleukin 1 and parathyroid hormone-related protein are thought to be involved in the development of high serum Ca(++) levels, its precise underlying mechanism remains unknown. This study analyzed the expression of various genes that are thought to regulate serum Ca(++) levels in ATL and showed that the overexpression of the receptor activator of nuclear factor kappaB (RANK) ligand gene correlated with hypercalcemia. ATL cells from patients with hypercalcemia, which highly expressed the transcripts of the RANK ligand (RANKL) gene, induced the differentiation of human hematopoietic precursor cells (HPCs) into OCLs in vitro in the presence of macrophage colony-stimulating factor (M-CSF). In contrast, ATL cells from patients without hypercalcemia did not induce such differentiation, suggesting that the induction of the differentiation correlated with the expression of the RANKL gene in ATL cells. Cell differentiation was suppressed by osteoprotegerin/Fc, an inhibitor of RANKL, indicating that such differentiation occurred through the RANK-RANKL pathway. In addition, direct contact between ATL cells and HPCs was essential for the differentiation, suggesting that not the soluble form but membrane-bound RANKL played a role in this process. These results strongly suggested that ATL cells induce the differentiation of HPCs to OCLs through RANKL expressed on their surface, in cooperation with M-CSF, and ultimately cause hypercalcemia.

Topics: Adult; Aged; Bone Resorption; Carrier Proteins; Cell Differentiation; Female; Gene Expression Regulation, Leukemic; Glycoproteins; Humans; Hypercalcemia; Immunophenotyping; Leukemia-Lymphoma, Adult T-Cell; Lymphocyte Activation; Macrophage Colony-Stimulating Factor; Male; Membrane Glycoproteins; Membrane Proteins; Middle Aged; Neoplasm Proteins; Neoplastic Stem Cells; Osteoclasts; Osteoprotegerin; Parathyroid Hormone-Related Protein; Protein Biosynthesis; Proteins; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Solubility; T-Lymphocytes

Prostaglandin E2 (PGE2) can stimulate bone resorption by a cyclic AMP-dependent pathway. Two PGE2 receptors, EP2 and EP4 have been shown to play a role in PGE2 stimulation of osteoclast formation. In primary osteoblastic cell cultures from EP2 wild type (EP2 +/+) mice, PGE2 (0.1 microM) increased cyclic AMP production 3.5-fold, but PGE2 had no effect on cells from mice in which the EP2 receptor had been deleted (EP2 -/-). To examine the role of the EP2 receptor in the resorption response in vivo we injected PGE2 in EP2 -/- mice, and compared them with EP2 +/+ mice. Injection of PGE2 (3 mg/kg, four times daily for three days) in 9- to 12-month-old male mice on a 129 SvEv background increased serum calcium from 9.8 +/- 0.5 to 10.7 +/- 0.3 mg/dl (P < 0.01) in EP2 +/+ mice but not in EP2 -/- mice (10.1 +/- 0.3 vs. 10.2 +/- 0.3 mg/dl). PGE2 injection (6 mg/kg twice a day for three days) in 3-4 month old male mice on a C57 BL/6 X 129 SvEv background increased calcium from 8.2 +/- 0.1 to 9.0 +/- 0.3 mg/dl (P < 0.05) in EP2 +/+ mice but had no effect in EP2-/- mice (8.4 +/- 0.1 vs. 8.3 +/- 0.2 mg/dl). Injection of PGE2 over the calvariae of EP2 +/+ and EP2-/- mice increased the expression of receptor activator of nuclear factor kappaB ligand (RANKL) both locally and in the tibia, but RANKL responses were lower in EP2 -/- mice. We conclude that EP2 receptor plays a role in the hypercalcemic response to PGE2. This impaired response in EP2 -/- mice may be due to decreased ability to stimulate cyclic AMP and in part, to a smaller increase in the expression of RANKL mRNA.

Topics: Actins; Adenylyl Cyclases; Animals; Blotting, Northern; Bone and Bones; Calcium; Carrier Proteins; Cyclic AMP; Dinoprostone; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); Glycoproteins; Hypercalcemia; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Osteoblasts; Osteoclasts; Osteogenesis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP2 Subtype; Receptors, Tumor Necrosis Factor; RNA, Messenger

Osteoprotegerin (OPG), a novel, secreted tumor necrosis factor receptor family member that inhibits osteoclast formation and activity was examined for its activity in a syngeneic tumor model of humoral hypercalcemia of malignancy. Normal mice bearing Colon-26 tumors develop increases in both parathyroid hormone-related protein (PTHrP) expression and plasma PTHrP, marked hypercalcemia, and increased bone resorption. OPG, given either at the onset of hypercalcemia or after it had occurred, blocked tumor-induced increases in bone resorption and hypercalcemia and rapidly normalized blood ionized calcium. In tumor-bearing mice, OPG treatments reduced osteoclast activity from approximately 2-fold above normal into the subphysiological range but had no effects on tumor size, tumor-induced cachexia, or PTHrP levels. The potent effects of OPG in this humoral hypercalcemia of malignancy model suggest a potential therapeutic role for OPG in the prevention and treatment of this disorder.

Topics: Animals; Dose-Response Relationship, Drug; Glycoproteins; Hypercalcemia; Male; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Neoplasms, Experimental; Osteoclasts; Osteoprotegerin; Parathyroid Hormone-Related Protein; Proteins; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Osteoprotegerin (OPG) and OPG-ligand (OPGL) potently inhibit and stimulate, respectively, osteoclast differentiation (Simonet, W.S., D.L. Lacey, C.R. Dunstan, M. Kelley, M.-S. Chang, R. Luethy, H.Q. Nguyen, S. Wooden, L. Bennett, T. Boone, et al. 1997. Cell. 89:309-319; Lacey, D.L., E. Timms, H.-L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, et al. 1998. Cell. 93: 165-176), but their effects on mature osteoclasts are not well understood. Using primary cultures of rat osteoclasts on bone slices, we find that OPGL causes approximately sevenfold increase in total bone surface erosion. By scanning electron microscopy, OPGL-treated osteoclasts generate more clusters of lacunae on bone suggesting that multiple, spatially associated cycles of resorption have occurred. However, the size of individual resorption events are unchanged by OPGL treatment. Mechanistically, OPGL binds specifically to mature OCs and rapidly (within 30 min) induces actin ring formation; a marked cytoskeletal rearrangement that necessarily precedes bone resorption. Furthermore, we show that antibodies raised against the OPGL receptor, RANK, also induce actin ring formation. OPGL-treated mice exhibit increases in blood ionized Ca++ within 1 h after injections, consistent with immediate OC activation in vivo. Finally, we find that OPG blocks OPGL's effects on both actin ring formation and bone resorption. Together, these findings indicate that, in addition to their effects on OC precursors, OPGL and OPG have profound and direct effects on mature OCs and indicate that the OC receptor, RANK, mediates OPGL's effects.

Topics: Actins; Animals; Bone and Bones; Bone Resorption; Carrier Proteins; Cattle; Cell Count; Cell Differentiation; Cells, Cultured; Cytoskeleton; Glycoproteins; Hematopoietic Stem Cells; Hypercalcemia; Membrane Glycoproteins; Mice; Mice, Inbred Strains; Microscopy, Electron, Scanning; Osteoclasts; Osteoporosis; Osteoprotegerin; RANK Ligand; Rats; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor

Osteoprotegerin (OPG) is a secreted protein that inhibits osteoclast formation and activity and appears to be a critical regulator of bone mass and metabolism. In the current study, mice were challenged with various cytokines and hormones (interleukin-1beta, tumor necrosis factor-alpha, parathyroid hormone, parathyroid hormone-related protein, and 1alpha,25-dihydroxyvitamin D3) that are known to increase bone resorption and cause hypercalcemia and treated concurrently with either a recombinant chimeric Fc fusion form of human OPG, with enhanced biological activity (cOPG) (2.5 mg/kg/day) or vehicle. Mice receiving these bone-resorbing factors became hypercalcemic by day 3 after commencing treatment and had increased bone resorption as evidenced by elevated osteoclast numbers on day 5. Concurrent cOPG treatment prevented hypercalcemia (p < 0.05) and maintained osteoclast numbers in the normal range (p < 0.001). The demonstration that cOPG can inhibit bone resorption suggests that this molecule may be useful in the treatment of diseases including hyperparathyroidism, humoral hypercalcemia of malignancy, osteoporosis, and inflammatory bone disease, which are characterized, in part, by increases in osteoclastic bone resorption.

Topics: Animals; Bone and Bones; Bone Resorption; Calcitriol; Calcium; Cell Count; Glycoproteins; Humans; Hypercalcemia; Interleukin-1; Isomerism; Male; Mice; Osteoclasts; Osteoprotegerin; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Proteins; Radiography; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tumor Necrosis Factor-alpha; Weight Loss

The ligand for osteoprotegerin has been identified, and it is a TNF-related cytokine that replaces the requirement for stromal cells, vitamin D3, and glucocorticoids in the coculture model of in vitro osteoclastogenesis. OPG ligand (OPGL) binds to a unique hematopoeitic progenitor cell that is committed to the osteoclast lineage and stimulates the rapid induction of genes that typify osteoclast development. OPGL directly activates isolated mature osteoclasts in vitro, and short-term administration into normal adult mice results in osteoclast activation associated with systemic hypercalcemia. These data suggest that OPGL is an osteoclast differentiation and activation factor. The effects of OPGL are blocked in vitro and in vivo by OPG, suggesting that OPGL and OPG are key extracellular regulators of osteoclast development.

Topics: Amino Acid Sequence; Animals; Bone Resorption; Carrier Proteins; Cell Differentiation; Cells, Cultured; Cloning, Molecular; Coculture Techniques; Cytokines; Gene Expression Regulation, Developmental; Glycoproteins; Hematopoietic Stem Cells; Humans; Hypercalcemia; Ligands; Macrophage Colony-Stimulating Factor; Membrane Glycoproteins; Mice; Molecular Sequence Data; Organ Specificity; Osteoclasts; Osteoprotegerin; Protein Binding; RANK Ligand; Rats; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Fusion Proteins; RNA, Messenger

Osteoclastogenesis inhibitory factor (OCIF) is a novel secreted protein that inhibits osteoclastogenesis both in vitro and in vivo. In this study, we examined the effects of OCIF on serum calcium (Ca) concentrations in normal mice and in hypercalcemic nude mice carrying tumors associated with humoral hypercalcemia of malignancy. In normal mice, a single intraperitoneal injection of OCIF reduced serum Ca levels in a dose-dependent manner. Significant decrease in serum Ca (by 1.6 +/- 0.3 mg/dL, n = 5) was observed 2 h after the injection of OCIF at 20 mg/kg and the hypocalcemic effect continued for up to 12 h. Serum phosphate (Pi) concentrations also decreased in response to OCIF. Urinary excretion of Ca, Pi, and creatinine did not change significantly after injection of OCIF or vehicle. In hypercalcemic, tumor-bearing nude mice, a single intraperitoneal injection of OCIF at 20 mg/kg resulted in a dramatic decrease in serum Ca (maximal decrease 2.8 +/- 0.37 mg/dL, n = 11), which continued for up to 24 h. The results suggest that OCIF decreased serum Ca through its inhibitory effect on bone resorption. Furthermore, it is suggested that OCIF has therapeutic potential for the treatment of hypercalcemic conditions such as malignancy-associated hypercalcemia.

Topics: Animals; Calcium; Creatinine; Disease Models, Animal; Dose-Response Relationship, Drug; Glycoproteins; Hypercalcemia; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Proteins; Neoplasms, Experimental; Osteoprotegerin; Parathyroid Hormone-Related Protein; Phosphates; Proteins; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Transforming Growth Factor alpha