In vivo impact of a 4 bp deletion mutation in the DLX3 gene on bone development.

Distal-less 3 (DLX3) gene mutations are etiologic for Tricho-Dento-Osseous syndrome. To investigate the in vivo impact of mutant DLX3 on bone development, we established transgenic (TG) mice expressing the c.571_574delGGGG DLX-3 gene mutation (MT-DLX3) driven by a mouse 2.3 Col1A1 promoter. Microcomputed tomographic analyses demonstrated markedly increased trabecular bone volume and bone mineral density in femora from TG mice. In ex vivo experiments, TG mice showed enhanced differentiation of bone marrow stromal cells to osteoblasts and increased expression levels of bone formation markers. However, TG mice did not show enhanced dynamic bone formation rates in in vivo fluorochrome double labeling experiments. Osteoclastic differentiation capacities of bone marrow monocytes were reduced in TG mice in the presence of osteoclastogenic factors and the numbers of TRAP(+) osteoclasts on distal metaphyseal trabecular bone surfaces were significantly decreased. TRACP 5b and CTX serum levels were significantly decreased in TG mice, while IFN-gamma levels were significantly increased. These data demonstrate that increased levels of IFN-gamma decrease osteoclast bone resorption activities, contributing to the enhanced trabecular bone volume and mineral density in these TG mice. These data suggest a novel role for this DLX-3 mutation in osteoclast differentiation and bone resorption.

Identification and characterization of osteoclast-like cells and their progenitors in cultures of feline marrow mononuclear cells.

The predominant cell responsible for bone resorption, the multinucleated osteoclast, has been difficult to study because of inaccessibility. When feline marrow-derived mononuclear cells are established in long-term culture, multinucleated cells form within 48 h, reaching maximum numbers at 16 d. We have observed that these cultured cells have many of the features of osteoclasts. Morphologically, they are multinucleated, contain large numbers of branched mitochondria, have a peripheral cytoplasm lacking organelles (a clear zone), and have extensive cell-surface processes. In addition to these ultrastructural features, the cells contain a tartrate-resistant acid phosphatase, the activity of which is increased by parathyroid hormone (PTH) and inhibited by calcitonin. PTH, prostaglandin E2, and 1,25(OH)2 vitamin D3 increased multinucleated cell formation, while calcitonin inhibited the stimulatory effects of PTH. Time-lapse cinemicrographic and autoradiographic studies indicated that the multinucleated cells formed by fusion of the mononuclear progenitors. The multinucleated cells were phagocytic and stained with nonspecific esterase, consistent with their being derived from immature monocytes. Further, cell populations enriched for multinucleated cells release 45Ca from devitalized bone. Density-gradient centrifugation on Percoll was used to enrich and characterize the mononuclear progenitors of these multinucleated cells. The progenitor cells were found predominantly in Percoll density layers of 1.065 to 1.08 g/ml and were enriched up to 30-fold as compared to unfractionated cells. The bone marrow mononuclear cells that formed the multinucleated cells were initially nonadherent to plastic, stained heavily with nonspecific esterase, and appeared to be immature monocytes histologically. These data indicate that the multinucleated osteoclast-like cells in our cultures are derived from nonadherent monocytic progenitor cells that are responsive to osteotropic hormones. The ability to grow and characterize these cells in vitro should facilitate studies to elucidate the role these cells play in normal and pathologic states of bone resorption.

Involvement of hepcidin in the anemia of multiple myeloma.

PURPOSE: Hepcidin is a liver-produced peptide implicated in the anemia of inflammation. Because interleukin (IL)-6 is a potent inducer of hepcidin expression and its levels are elevated in multiple myeloma, we studied the role of hepcidin in the anemia of multiple myeloma. EXPERIMENTAL DESIGN: Urinary hepcidin and serum levels of IL-6, ferritin, C-reactive protein, tumor necrosis factor-alpha, and IL-1 beta were studied in newly diagnosed myeloma patients. In vitro hepcidin induction assay was assessed by real-time PCR assay. RESULTS: Pretreatment urinary hepcidin levels in 44 patients with stage III multiple myeloma were 3-fold greater than normal controls. In the subset of multiple myeloma patients without renal insufficiency (n = 27), a marked inverse correlation was seen between hemoglobin at diagnosis and urinary hepcidin level (P = 0.014) strongly supporting a causal relationship between up-regulated hepcidin expression and anemia. The urinary hepcidin also significantly (P < 0.05) correlated with serum ferritin and C-reactive protein, whereas its correlation with serum IL-6 levels was of borderline significance (P = 0.06). Sera from 14 multiple myeloma patients, with known elevated urinary hepcidin, significantly induced hepcidin mRNA in the Hep3B cells, whereas normal sera had no effect. For 10 patients, the ability of anti-IL-6 and anti-IL-6 receptor antibodies to prevent the serum-induced hepcidin RNA was tested. In 6 of these patients, hepcidin induction was abrogated by the anti-IL-6 antibodies, but in the other 4 patients, the neutralizing antibodies had no effect. CONCLUSIONS: These results indicate hepcidin is up-regulated in multiple myeloma patients by both IL-6-dependent and IL-6-independent mechanisms and may play a role in the anemia of multiple myeloma.

Advances in bone biology: the osteoclast.

Much has been learned about the cell biology and molecular biology of the osteoclast in the last 5 yr. The osteoclast appears to be derived from CFU-GM, the committed monocyte-granulocyte precursor cell. This cell then differentiates into more committed precursors for the osteoclast. The role of the marrow microenvironment appears to be critical for murine osteoclast formation, although in human systems it appears to be nonessential but acts to enhance osteoclast formation and resorption. The osteoclast has been shown to be a secretory cell capable of producing both stimulators and inhibitors of osteoclast formation and resorption. The identification of the role of protooncogenes, such as c-fos and pp60c-src, in osteoclast differentiation and bone resorption has provided important insights into the regulation of normal osteoclast activity. Studies such as these should help us to dissect the pathophysiology of abnormal osteoclastic activity, such as seen in hypercalcemia of malignancy, osteopetrosis, and Paget's disease of bone. Future research is needed to further delineate the signaling pathways involved in osteoclastic bone resorption in response to cytokines and hormones, as well as to identify the molecular events required for commitment of multipotent precursors to the osteoclast lineage. Development of osteoclast cell lines may be possible and would greatly enhance our understanding of the biology of the osteoclast. Utilization of current model systems to examine the effects of cytokines and hormones on osteoclast precursors in vitro and in vivo and the ability to obtain large numbers of highly purified osteoclasts for production of osteoclast cDNA libraries should lead to important new discoveries in osteoclast biology.

Downregulation of calcitonin receptor mRNA expression by calcitonin during human osteoclast-like cell differentiation.

Calcitonin inhibits both osteoclast formation and bone resorption, and is a primary treatment for patients with hypercalcemia and increased bone turnover. However, the clinical utility of calcitonin is limited because patients become refractory to calcitonin after several days (the calcitonin "escape phenomenon"). The molecular basis for calcitonin "escape" is unclear. To determine the regulatory mechanisms controlling calcitonin receptor (CTR) expression in osteoclasts and their precursors, we treated immature mononuclear precursors for human osteoclast-like multinucleated cells (MNC) formed in vitro with 1,25-(OH)2D3, to induce their differentiation to committed mononuclear precursors, and mature multinucleated osteoclasts, and used reverse transcriptase (RT)-PCR to assess expression of CTR mRNA in both committed mononuclear precursors and MNC. The PCR fragment produced was cloned and sequenced to confirm that it was derived from CTR mRNA. CTR mRNA expression was detected in mononuclear MNC precursors after 7 d of 1,25-(OH)2D3 treatment. It was also present in osteoclast-like MNC and highly purified giant cells from osteoclastomas, but not in monocytes or macrophage polykaryons formed in vitro. Calcitonin markedly decreased CTR but not actin mRNA expression in giant cells and MNC after 12 h, and removal of calcitonin restored CTR mRNA expression. Similarly, calcitonin decreased calcitonin-induced adenylate cyclase activity. These data suggest: (a) downregulation of CTR gene expression by calcitonin may in part explain the calcitonin "escape phenomenon"; and (b) expression of CTR mRNA occurs in mononuclear osteoclast precursors within 7 d after exposure to 1,25-(OH)2D3.

Osteoclasts expressing the measles virus nucleocapsid gene display a pagetic phenotype.

Osteoclasts (OCLs) in Paget's disease are markedly increased in number and size, have increased numbers of nuclei per multinucleated cell, and demonstrate increased resorption capacity and increased sensitivity to 1,25-(OH)(2)D(3), the active form of vitamin D. These cells also contain nuclear inclusions, reminiscent of those seen in paramyxovirus-infected cells, which cross-react with antibodies to measles virus nucleocapsid (MVNP) antigen. To elucidate the role of MV in the abnormal OCL phenotype of Paget's disease, we transduced normal OCL precursors with retroviral vectors expressing MVNP and the MV matrix (MVM) genes. The transduced cells were then cultured with 1,25-(OH)(2)D(3) for14 or 21 days to induce formation of OCL-like multinucleated cells. The MVNP-transduced cells formed increased numbers of multinucleated cells, which contained many more nuclei and had increased resorption capacity compared with multinucleated cells derived from empty vector-transduced (EV-transduced) and MVM-transduced or normal bone marrow cells. Furthermore, MVNP-transduced cells showed increased sensitivity to 1, 25-(OH)(2)D(3), and formed OCLs at concentrations of 1, 25-(OH)(2)D(3) that were 1 log lower than that required for normal, EV-transduced, or MVM-transduced cells. These results demonstrate that expression of the MVNP gene in normal OCL precursors stimulates OCL formation and induces OCLs that express a phenotype similar to that of pagetic OCLs. These results support a potential pathophysiologic role for MV infection in the abnormal OCL activity and morphology that are characteristic of pagetic OCLs.

Atypical multinucleated cells form in long-term marrow cultures from patients with Paget's disease.

Although Paget's disease is the most flagrant example of a primary osteoclast disorder, little is known of osteoclast biology in this disease. In this report we have studied the formation of cells with the osteoclast phenotype in long-term cultures of marrow mononuclear cells derived from patients with Paget's disease, and compared these with similar cells formed in long-term marrow cultures from normal individuals, and with osteoclasts present in pagetic bone. Osteoclasts formed in pagetic marrow cultures resembled osteoclasts present in pagetic bone, but were distinctly different from osteoclasts formed in normal marrow cultures. Osteoclast formation was 10-20-fold greater in pagetic marrow cultures than in normal cultures. The multinucleated cells formed in cultures of pagetic marrow were much larger in size, were hyperresponsive to 1,25(OH)2 vitamin D, had more nuclei per cell, had increased levels of tartrate-resistant acid phosphatase activity and had ultrastructural features which were not seen in multinucleated cells formed from normal marrow mononuclear cells. These pagetic marrow-derived multinucleated cells formed large resorption lacunae on calcified matrices and cross-reacted with monoclonal antibodies which preferentially bind to osteoclasts. The multinucleated cells formed from marrow obtained from uninvolved sites in Paget's patients also displayed these abnormal features.

Recombinant human transforming growth factor-alpha stimulates the formation of osteoclast-like cells in long-term human marrow cultures.

Transforming growth factor-alpha (TGF-alpha) is synthesized by a variety of tumor cell lines and stimulates osteoclastic bone resorption in vitro. The mechanism by which TGF-alpha increases osteoclast activity is unknown. We used a human marrow culture system that forms osteoclast-like multinucleated cells (MNCs) to determine the effects of recombinant human TGF-alpha on MNC formation. Addition of 0.01 ng/ml TGF-alpha for the 1st week followed by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] for the subsequent 2 wk significantly increased MNCs. Treatment of these cultures with TGF-alpha without later addition of 1,25(OH)2D3 did not increase MNC formation. Autoradiographic studies revealed that TGF-alpha stimulated proliferation of precursors for MNCs, and 1,25(OH)2D3 increased their rate of fusion into MNCs. Addition of murine epidermal growth factor (EGF) (0.1 ng/ml) followed by 1,25(OH)2D3 also significantly stimulated MNC formation. These data suggest that TGF-alpha and EGF may stimulate bone resorption by increasing the proliferation of osteoclast precursors, which leads to increased numbers of osteoclasts.

Interleukin 6. A potential autocrine/paracrine factor in Paget's disease of bone.

Pagetic osteoclasts are greatly increased in number and size and have increased numbers of nuclei per cell compared to normal osteoclasts. The mechanisms responsible for enhanced osteoclast formation in Paget's disease are unknown. We have used our recently described model system for pagetic osteoclast formation to evaluate culture media conditioned by these atypical multinucleated cells (MNC) to determine if pagetic osteoclasts produce an autocrine or paracrine factor that enhances osteoclast formation. Conditioned media from long-term bone marrow cultures from patients with Paget's disease stimulated osteoclast-like MNC formation in normal marrow cultures. At least part of this activity could be ascribed to interleukin 6 (IL-6). In contrast, conditioned media from normal marrow cultures contained lower levels of IL-6 and did not stimulate formation of osteoclast-like MNC. 7 of 8 bone marrow plasma samples taken from involved bones and 18 of 27 peripheral blood serum samples from Paget's patients had high levels of IL-6. Normal marrow plasma and peripheral blood serum had no or very low levels of IL-6. These results suggest that IL-6 produced by marrow and/or bone cells in patients with Paget's disease may be an autocrine/paracrine factor for pagetic osteoclasts.

Cloning and identification of human Sca as a novel inhibitor of osteoclast formation and bone resorption.

Increased osteoclast activity is responsible for the enhanced bone destruction in postmenopausal osteoporosis, Paget's disease, bone metastasis, and hypercalcemia of malignancy. However, the number of known inhibitory factors that block osteoclast formation and bone resorption are limited. Therefore, we used an expression-cloning approach to identify novel factors produced by osteoclasts that inhibit osteoclast activity. A candidate clone was identified and isolated from a human osteoclast-like multinucleated cell (MNC) cDNA library, named osteoclast inhibitory peptide-1 (OIP-1), and the cDNA sequence was determined. This sequence matched that of the recently identified human stem cell antigen, was structurally similar to the mouse Ly-6 gene family, and the sequence predicted it was a glycosyl phosphatidyl inositol (GPI)-anchored protein that had a cleavable COOH-terminal peptide. Western blot analysis of conditioned media from 293 cells transfected with the OIP-1 cDNA clone confirmed that OIP-1 was released into the media as a membrane-bound GPI-linked protein. Interestingly, both recombinant OIP-1 expressed in Escherichia coli (which does not have GPI linker) and OIP-1 expressed by mammalian cells significantly reduced osteoclast-like MNC formation induced by 1,25-dihydroxyvitamin D3 or PTH-related protein in mouse and human bone marrow cultures, and inhibited 45Ca release from prelabeled bone in fetal rat organ cultures. In contrast, recombinant OIP-1 did not inhibit the growth of a variety of other cell types. These data indicate that OIP-1 is a novel, specific inhibitor of osteoclast formation and bone resorption.

Annexin II increases osteoclast formation by stimulating the proliferation of osteoclast precursors in human marrow cultures.

Annexin II (AXII), a calcium-dependent phospholipid-binding protein, has been recently found to be an osteoclast (OCL) stimulatory factor that is also secreted by OCLs. In vitro studies showed that AXII induced OCL formation and bone resorption. However, the mechanism of action by which AXII acts as a soluble extracellular protein to induce OCL formation is unknown. In this paper, we demonstrate that AXII gene expression is upregulated by 1,25-dihydroxyvitamin D3 [1, 25-(OH)2D3] and that addition of AXII significantly increased OCL-like multinucleated cell formation. Time-course studies suggested that AXII acted on the proliferative stage of OCL precursors and that AXII increased thymidine incorporation in OCL precursors. Moreover, AXII enhanced the growth of CFU-GM, the earliest identifiable OCL precursor, when bone marrow cultures were treated with low concentrations of GM-CSF. This capacity of AXII to induce OCL precursor proliferation was due to induction of GM-CSF expression, because the addition of neutralizing antibodies to GM-CSF blocked the stimulatory effect of AXII on OCL formation. RT-PCR analysis using RNA from highly purified subpopulations of marrow cells demonstrated that T cells, especially CD4(+) T cells, produced GM-CSF in response to AXII. Furthermore, FACS(R) analysis of T-cell subpopulations treated with fluorescein-labeled AXII suggested that the CD4(+), but not CD8(+), subpopulation of T cells express an AXII receptor. Taken together, these data suggest that AXII stimulates OCL formation by activating T cells through a putative receptor to secrete GM-CSF. GM-CSF then expands the OCL precursor pool to enhance OCL formation.

Antisense inhibition of macrophage inflammatory protein 1-alpha blocks bone destruction in a model of myeloma bone disease.

We recently identified macrophage inflammatory protein 1-alpha (MIP-1alpha) as a factor produced by multiple myeloma (MM) cells that may be responsible for the bone destruction in MM (1). To investigate the role of MIP-1alpha in MM bone disease in vivo, the human MM-derived cell line ARH was stably transfected with an antisense construct to MIP-1alpha (AS-ARH) and tested for its capacity to induce MM bone disease in SCID mice. Human MIP-1alpha levels in marrow plasma from AS-ARH mice were markedly decreased compared with controls treated with ARH cells transfected with empty vector (EV-ARH). Mice treated with AS-ARH cells lived longer than controls and, unlike the controls, they showed no radiologically identifiable lytic lesions. Histomorphometric analysis demonstrated that osteoclasts (OCLs) per square millimeter of bone and OCLs per millimeter of bone surface of AS-ARH mice were significantly less than in EV-ARH mice, and the percentage of tumors per total bone area was also significantly decreased. AS-ARH cells demonstrated decreased adherence to marrow stromal cells, due to reduced expression of the alpha(5)beta(1) integrin and diminished homing capacity and survival. These data support an important role for MIP-1alpha in cell homing, survival, and bone destruction in MM.

Interleukin-6 enhances hypercalcemia and bone resorption mediated by parathyroid hormone-related protein in vivo.

Tumors frequently induce the multifunctional cytokine IL-6, which has been linked to several paraneoplastic syndromes, most notably cachexia. IL-6 stimulates osteoclast formation, causes mild hypercalcemia, and is produced by bone cells in vitro upon exposure to systemic hormones. Since IL-6 is produced together with parathyroid hormone-related protein (PTH-rP) in some patients with cancer, we tested the hypothesis that production of IL-6 potentiates the effects of PTH-rP on Ca2+ homeostasis and osteoclastic bone resorption and examined potential mechanisms for these interactions in vivo. Chinese hamster ovarian (CHO) cells stably transfected with cDNAs for IL-6 (CHO/IL-6) and PTH-rP sense (CHO/PTH-rP) or antisense (CHO/PTH-rP AS) were inoculated intramuscularly into nude mice. Experimental groups included CHO/IL-6 plus CHO/PTH-rP; CHO/IL-6 plus CHO/PTH-rP AS; CHO/IL-6 alone; and CHO/PTH-rP alone. Blood ionized Ca2+ was measured on days 0, 7, 10, 12, and 13. Three different developmental stages in the osteoclast lineage were examined at day 13: the early multipotential precursor, granulocyte macrophage colony-forming units (CFU-GM); more mature mononuclear osteoclast precursors, assessed by their capacity to form tartrate-resistant acid phosphatase-positive multinucleated cells in marrow cultures; and mature osteoclasts, assessed by histomorphometry. IL-6 increased CFU-GM but not bone resorption or Ca2+. In contrast, PTH-rP induced hypercalcemia and bone resorption and increased multinucleated osteoclasts and more mature precursors cells, but not CFU-GM. However, mice treated with both IL-6 and PTH-rP had very marked hypercalcemia and osteoclastosis as well as an increase in the number of both CFU-GM and mature osteoclast precursors. These data demonstrate that IL-6 enhances PTH-rP-mediated hypercalcemia and bone resorption, most likely by increasing the pool of early osteoclast precursors that in turn can differentiate to mature osteoclasts. We conclude that IL-6 stimulatory effects on osteoclast precursors may enhance the effects of other bone resorption factors that act at later stages in the osteoclast lineage.

Enhanced RANK ligand expression and responsivity of bone marrow cells in Paget's disease of bone.

Paget's disease is characterized by highly localized areas of increased osteoclast (OCL) activity. This suggests that the microenvironment in pagetic lesions is highly osteoclastogenic, or that OCL precursors in these lesions are hyperresponsive to osteoclastogenic factors (or both). To examine these possibilities, we compared RANK ligand (RANKL) mRNA expression in a marrow stromal cell line developed from a pagetic lesion (PSV10) with that in a normal stromal cell line (Saka), and expression in marrow samples from affected bones of Paget's patients with that in normal marrow. RANKL mRNA was increased in PSV10 cells and pagetic marrow compared with Saka cells and normal marrow, and was also increased in marrow from affected bones compared with uninvolved bones from Paget's patients. Furthermore, pagetic marrow cells formed OCLs at much lower RANKL concentrations than did normal marrow. Anti-IL-6 decreased the RANKL responsivity of pagetic marrow to normal levels, whereas addition of IL-6 to normal marrow enhanced RANKL responsivity. Thus, RANKL expression and responsivity is increased in pagetic lesions, in part mediated by IL-6. These data suggest that the combination of enhanced expression of RANKL in affected bones and increased RANKL sensitivity of pagetic OCL precursors may contribute to the elevated numbers of OCLs in Paget's disease.

Osteoclast-like cells form in long-term human bone marrow but not in peripheral blood cultures.

Transplantation studies have suggested that peripheral blood mononuclear cells contain precursors for osteoclasts. Thus we tested the capacity of peripheral blood monocytes to form osteoclasts in long-term culture. We have reported previously that mononuclear cells from feline, baboon, and human marrow form osteoclast-like cells in long term cultures. Further, the formation of these cells is increased in response to bone resorption stimulatory agents such as PTH, interleukin 1, and transforming growth factor alpha. We now report that these cells show characteristic cytoplasmic contraction with calcitonin and form resorption lacunae when cultured on sperm whale dentine. Thus, these bone marrow-derived multinucleated cells fulfill the functional criteria for osteoclasts. Although cultured peripheral blood monocytes can be induced to form multinucleated cells with 1,25-dihydroxyvitamin D3, these cells did not show similar responses to the osteotropic factors as multinucleated cells formed in the bone marrow cultures multinucleated cells. These results indicate that osteoclasts or cells closely related to osteoclasts form in long-term human bone marrow cultures. In contrast, few mononuclear cells in the peripheral blood appear capable of forming osteoclasts under the culture conditions used in these experiments.

Immortalization of osteoclast precursors by targeting Bcl -XL and Simian virus 40 large T antigen to the osteoclast lineage in transgenic mice.

Cellular and molecular characterization of osteoclasts (OCL) has been extremely difficult since OCL are rare cells, and are difficult to isolate in large numbers. We used the tartrate-resistant acid phosphatase promoter to target the bcl-XL and/or Simian Virus 40 large T antigen (Tag) genes to cells in the OCL lineage in transgenic mice as a means of immortalizing OCL precursors. Immunocytochemical studies confirmed that we had targeted Bcl-XL and/or Tag to OCL, and transformed and mitotic OCL were readily apparent in bones from both Tag and bcl-XL/Tag mice. OCL formation in primary bone marrow cultures from bcl-XL, Tag, or bcl-XL/Tag mice was twofold greater compared with that of nontransgenic littermates. Bone marrow cells from bcl-XL/Tag mice, but not from singly transgenic bcl-XL or Tag mice, have survived in continuous culture for more than a year. These cells form high numbers of bone-resorbing OCL when cultured using standard conditions for inducing OCL formation, with approximately 50% of the mononuclear cells incorporated into OCL. The OCL that form express calcitonin receptors and contract in response to calcitonin. Studies examining the proliferative capacity and the resistance of OCL precursors from these transgenic mice to apoptosis demonstrated that the increased numbers of OCL precursors in marrow from bcl-XL/Tag mice was due to their increased survival rather than an increased proliferative capacity compared with Tag, bcl-XL, or normal mice. Histomorphometric studies of bones from bcl-XL/Tag mice also confirmed that there were increased numbers of OCL precursors (TRAP + mononuclear cells) present in vivo. These data demonstrate that by targeting both bcl-XL and Tag to cells in the OCL lineage, we have immortalized OCL precursors that form bone-resorbing OCL with an efficiency that is 300-500 times greater than that of normal marrow.

Treatment strategies for bone disease.

Multiple myeloma is characterized by extensive bone destruction with little or no new bone formation. A multiplicity of factors including receptor activator NF-kappaB (RANKL), macrophage inflammatory protein-1alpha, interleukin-3 and interleukin-6 can induce osteoclast formation in myeloma and drive the bone destructive process. Furthermore, factors are also produced either in the microenvironment or by myeloma cells themselves, which inhibit osteoblast differentiation and new bone formation. The combination of increased osteoclast formation with little or no bone repair in response to the previous bone destruction explains the severity of the bone disease in myeloma. Studies of the pathophysiology of myeloma bone disease have identified several novel therapeutic targets. These include antibodies to RANKL, chemokine receptor antagonists, which block the effects of chemokines on osteoclast differentiation and proteasome antagonists, which can affect both RANKL production and osteoprotegerin levels as well as inhibit osteoclast and enhance osteoblast differentiation. In addition, many of the new biologic agents being used for the treatment of patients with myeloma also further inhibit the bone destructive process. New therapies that can target both the tumor as well as the severe bone disease should be on the horizon to treat this devastating complication of myeloma.

Genetic deletion of Sost or pharmacological inhibition of sclerostin prevent multiple myeloma-induced bone disease without affecting tumor growth.

Multiple myeloma (MM) causes lytic bone lesions due to increased bone resorption and concomitant marked suppression of bone formation. Sclerostin (Scl), an osteocyte-derived inhibitor of Wnt/ß-catenin signaling, is elevated in MM patient sera and increased in osteocytes in MM-bearing mice. We show here that genetic deletion of Sost, the gene encoding Scl, prevented MM-induced bone disease in an immune-deficient mouse model of early MM, and that administration of anti-Scl antibody (Scl-Ab) increased bone mass and decreases osteolysis in immune-competent mice with established MM. Sost/Scl inhibition increased osteoblast numbers, stimulated new bone formation and decreased osteoclast number in MM-colonized bone. Further, Sost/Scl inhibition did not affect tumor growth in vivo or anti-myeloma drug efficacy in vitro. These results identify the osteocyte as a major contributor to the deleterious effects of MM in bone and osteocyte-derived Scl as a promising target for the treatment of established MM-induced bone disease. Further, Scl did not interfere with efficacy of chemotherapy for MM, suggesting that combined treatment with anti-myeloma drugs and Scl-Ab should effectively control MM growth and bone disease, providing new avenues to effectively control MM and bone disease in patients with active MM.

Tumor necrosis factor enhances parathyroid hormone-related protein-induced hypercalcemia and bone resorption without inhibiting bone formation in vivo.

Humoral hypercalcemia of malignancy results from the effects of tumor-produced factors on bone, kidney, and intestine that disrupt normal calcium homeostasis. Although parathyroid hormone-related protein (PTHrP) is a major mediator of the syndrome, tumors also produce other hypercalcemic factors, such as tumor necrosis factor (TNF), which may modulate the effects of PTHrP. It has been postulated that TNF may counteract the stimulatory effects of PTHrP on bone formation. To examine the effects of TNF on PTHrP-induced changes in calcium and bone metabolism, a murine tumor model of hypercalcemia was used. Nude mice were inoculated with Chinese hamster ovarian (CHO) cells expressing human TNF (CHO/TNF) or nontransfected CHO cells (CHO/-) and further treated with injections of human PTHrP(1-34) or vehicle. The effects of TNF, PTHrP, and the combination of the two factors on blood ionized calcium, osteoclast recruitment, and bone histomorphometry were evaluated. Mice bearing CHO/TNF tumors that were injected with PTHrP had significantly higher calcium concentrations, increased committed osteoclast progenitors, and mature osteoclasts as well as enhanced bone resorption compared with mice bearing CHO/TNF tumors injected with vehicle or those bearing CHO/- tumors injected with PTHrP or vehicle. A 2-fold increase in new woven bone formed in the calvaria at sites of previous bone resorption was observed in CHO/TNF mice treated with PTHrP. Bone formation rates in the vertebrae were similar in both CHO/- and CHO/TNF mice treated with PTHrP. These data demonstrate that the hypercalcemic effects of PTHrP are enhanced by TNF and that this effect is due to the increased production of committed osteoclast precursors with a subsequent increase in osteoclastic bone resorption. Furthermore, PTHrP caused a coupled increase in osteoclastic bone resorption and new bone formation that was not inhibited by TNF. These findings highlight the complex interactions that may occur between tumor-produced factors on bone that result in malignancy-associated hypercalcemia and suggest that TNF may not be responsible for the decreased bone formation seen in some patients with this condition.

Evaluating results from the multiple myeloma patient subset treated with denosumab or zoledronic acid in a randomized phase 3 trial.

In a phase 3 trial of denosumab vs zoledronic acid in patients (n=1776) with bone metastases and solid tumors or multiple myeloma, denosumab was superior to zoledronic acid for the primary end point of prevention of skeletal-related events. There was no difference in overall survival between the two groups; however, an ad hoc overall survival analysis in the multiple myeloma subset of patients (n=180) favored zoledronic acid (hazard ratio (HR) 2.26; 95% confidence interval (CI) 1.13-4.50; P=0.014). In the present analysis, we found imbalances between the groups with respect to baseline risk characteristics. HRs with two-sided 95% CIs were estimated using the Cox model. After adjustment in a covariate analysis, the CI crossed unity (HR 1.86; 95% CI 0.90-3.84; P=0.0954). Furthermore, we found a higher rate of early withdrawals for the reasons of lost to follow-up and withdrawal of consent in the zoledronic acid group; after accounting for these, the HR was 1.31 (95% CI 0.80-2.15; P=0.278). In conclusion, the survival results in multiple myeloma patients in this trial were confounded and will eventually be resolved by an ongoing phase 3 trial.