Activation of NF-kappa B Signaling Promotes Growth of Prostate Cancer Cells in Bone

Patients with advanced prostate cancer almost invariably develop osseous metastasis. Although many studies indicate that the activation of NF-κB signaling appears to be correlated with advanced cancer and promotes tumor metastasis by influencing tumor cell migration and angiogenesis, the influence of altered NF-κB signaling in prostate cancer cells within boney metastatic lesions is not clearly understood. While C4-2B and PC3 prostate cancer cells grow well in the bone, LNCaP cells are difficult to grow in murine bone following intraskeletal injection. Our studies show that when compared to LNCaP, NF-κB activity is significantly higher in C4-2B and PC3, and that the activation of NF-κB signaling in prostate cancer cells resulted in the increased expression of the osteoclast inducing genes PTHrP and RANKL. Further, conditioned medium derived from NF-κB activated LNCaP cells induce osteoclast differentiation. In addition, inactivation of NF-κB signaling in prostate cancer cells inhibited tumor formation in the bone, both in the osteolytic PC3 and osteoblastic/osteoclastic mixed C4-2B cells; while the activation of NF-κB signaling in LNCaP cells promoted tumor establishment and proliferation in the bone. The activation of NF-κB in LNCaP cells resulted in the formation of an osteoblastic/osteoclastic mixed tumor with increased osteoclasts surrounding the new formed bone, similar to metastases commonly seen in patients with prostate cancer. These results indicate that osteoclastic reaction is required even in the osteoblastic cancer cells and the activation of NF-κB signaling in prostate cancer cells increases osteoclastogenesis by up-regulating osteoclastogenic genes, thereby contributing to bone metastatic formation.     

Role of Wnts in prostate cancer bone metastases

Prostate cancer (CaP) is unique among all cancers in that when it metastasizes to bone, it typically forms osteoblastic lesions (characterized by increased bone production). CaP cells produce many factors, including Wnts that are implicated in tumor-induced osteoblastic activity. In this prospectus, we describe our research on Wnt and the CaP bone phenotype. Wnts are cysteine-rich glycoproteins that mediate bone development in the embryo and promote bone production in the adult. Wnts have been shown to have autocrine tumor effects, such as enhancing proliferation and protecting against apoptosis. In addition, we have recently identified that CaP-produced Wnts act in a paracrine fashion to induce osteoblastic activity in CaP bone metastases. In addition to Wnts, CaP cells express the soluble Wnt inhibitor dickkopf-1 (DKK-1). It appears that DKK-1 production occurs early in the development of skeletal metastases, which results in masking of osteogenic Wnts, thus favoring osteolysis at the metastatic site. As metastases progress, DKK-1 expression decreases allowing for unmasking of Wnt's osteoblastic activity and ultimately resulting in osteosclerosis at the metastatic site. We believe that DKK-1 is one of the switches that transitions the CaP bone metastasis activity from osteolytic to osteoblastic. Wnt/DKK-1 activity fits a model of CaP-induced bone remodeling occurring in a continuum composed of an osteolytic phase, mediated by receptor activator of NFkB ligand (RANKL), parathyroid hormone-related protein (PTHRP) and DKK-1; a transitional phase, where environmental alterations promote expression of osteoblastic factors (Wnts) and decreases osteolytic factors (i.e., DKK-1); and an osteoblastic phase, in which tumor growth-associated hypoxia results in production of vascular endothelial growth factor and endothelin-1, which have osteoblastic activity. This model suggests that targeting both osteolytic activity and osteoblastic activity will provide efficacy for therapy of CaP bone metastases.     

Cover Image,Volume 120, Number 11, November 2019

Skeletal tissue homeostasis is maintained via the balance of osteoclastic bone resorption and osteoblastic bone formation. Autophagy and apoptosis are essential for the maintenance of homeostasis and normal development in cells and tissues. We found that Bax-interacting factor 1 (Bif-1/Endophillin B1/SH3GLB1), involving in autophagy and apoptosis, was upregulated during osteoclastogenesis. Furthermore, mature osteoclasts expressed Bif-1 in the cytosol, particularly the perinuclear regions and podosome, suggesting that Bif-1 regulates osteoclastic bone resorption. Bif-1-deficient (Bif-1 ^−/−) mice showed increased trabecular bone volume and trabecular number. Histological analyses indicated that the osteoclast numbers increased in Bif-1 ^−/− mice. Consistent with the in vivo results, osteoclastogenesis induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) was accelerated in Bif-1 ^−/− mice without affecting RANKL-induced activation of RANK downstream signals, such as NF-κB and mitogen-activated protein kinases (MAPKs), CD115/RANK expression in osteoclast precursors, osteoclastic bone-resorbing activity and the survival rate. Unexpectedly, both the bone formation rate and osteoblast surface substantially increased in Bif-1 ^−/− mice. Treatment with β-glycerophosphate (β-GP) and ascorbic acid (A.A) enhanced osteoblastic differentiation and mineralization in Bif-1 ^−/− mice. Finally, bone marrow cells from Bif-1 ^−/− mice showed a significantly higher colony-forming efficacy by the treatment with or without β-GP and A.A than cells from wild-type (WT) mice, suggesting that cells from Bif-1 ^−/− mice had higher clonogenicity and self-renewal activity than those from WT mice. In summary, Bif-1 might regulate bone homeostasis by controlling the differentiation and function of both osteoclasts and osteoblasts (235 words).     

Pleiotropic activity of lysophosphatidic acid in bone metastasis

Bone is a common metastatic site for solid cancers. Bone homeostasis is tightly regulated by intimate cross-talks between osteoblast (bone forming cells) and osteoclasts (bone resorbing cells). Once in the bone microenvironment, metastatic cells do not alter bone directly but instead perturb the physiological balance of the bone remodeling process controlled by bone cells. Tumor cells produce growth factors and cytokines stimulating either osteoclast activity leading to osteolytic lesions or osteoblast function resulting in osteoblastic metastases. Growth factors, released from the resorbed bone matrix or throughout osteoblastic bone formation, sustain tumor growth. Therefore, bone metastases are the sites of vicious cycles wherein tumor growth and bone metabolism sustain each other. Lysophosphatidic acid (LPA) promotes the growth of primary tumors and metastatic dissemination of cancer cells. We have shown that by acting on cancer cells via the contribution of blood platelets and the LPA-producing enzyme Autotaxin (ATX), LPA promotes the progression of osteolytic bone metastases in animal models. In the light of recent reports it would appear that the role of LPA in the context of bone metastases is complex involving multiple sources of lipid combined with direct and indirect effects on target cells. This review will present our current knowledge on the LPA/ATX axis involvement in osteolytic and osteoblastic skeletal metastases and will discuss the potential activity of LPA upstream and downstream metastasis seeding of cancer cells to bone as well as its implication in cancer induced bone pain. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Notch signaling and ERK activation are important for the osteomimetic properties of prostate cancer bone metastatic cell lines

Prostate cancer bone metastases are characterized by their ability to induce osteoblastic lesions and local bone formation. It has been suggested that bone metastatic prostate cancer cells are osteomimetic and capable of expressing genes and proteins typically expressed by osteoblasts. The ability of preosteoblasts to differentiate and express osteoblastic genes depends on several pathways, including Notch and MAPK. Here we show that notch1 expression is increased 4-5 times in C4-2B and MDA PCa 2b cells (osteoblastic skeletal prostate metastatic cancer cell lines) when compared with nonskeletal metastatic cell lines (LNCaP and DU145). Notch1 ligand, dll1, is expressed only in C4-2B cells. Immunohistochemical studies demonstrate that Notch1 is present in both human clinical samples from prostate cancer bone metastases and the C4-2B cell line. To determine whether prostate cancer bone metastases respond to osteogenic induction similar to osteoblasts, C4-2B cells were cultured in osteogenic medium that promotes mineralization. C4-2B cells mineralize and express HES-1 (a downstream target of Notch), an effect that is completely inhibited by L-685,458, a Notch activity inhibitor. Furthermore, osteogenic induction increases ERK activation, runx2 expression, and nuclear localization, independent of Notch signaling. Finally, we show that Notch and ERK activation are essential for Runx2 DNA binding activity and osteocalcin gene expression in C4-2B cells in response to osteogenic induction. These studies demonstrate that prostate cancer bone metastatic cell lines acquire osteoblastic properties through independent activation of ERK and Notch signaling; presumably, both pathways are activated in the bone microenvironment.     

Prostate cancer promotes CD11b positive cells to differentiate into osteoclasts

Bone is the preferred site of prostate cancer metastasis, contributing to the morbidity and mortality of this disease. A key step in the successful establishment of prostate cancer bone metastases is activation of osteoclasts with subsequent bone resorption causing the release of several growth factors from the bone matrix. CD11b+ cells in bone marrow are enriched for osteoclast precursors. Conditioned media from prostate cancer PC‐3 cells induces CD11b+ cells from human peripheral blood to differentiate into functional osteoclasts with subsequent bone resorption. Analysis of PC‐3 conditioned media revealed high amounts of IL‐6 and IL‐8. CD11b+ cells were cultured with M‐CSF and RANKL, IL‐6, IL‐8, and CCL2, alone or in combination. All of these conditions induced osteoclast fusion, but cells cultured with M‐CSF, IL‐6, IL‐8, and CCL2 were capable of limited bone resorption. Co‐incubation with IL‐6 and IL‐8 and the RANK inhibitor, RANK‐Fc, failed to inhibit osteoclast fusion and bone resorption, suggesting a potential RANKL‐independent mechanism of functional osteoclast formation. This study demonstrates that functional osteoclasts can be derived from CD11b+ cells derived from human PBMCs. Prostate cancer cells secrete factors, including IL‐6 and IL‐8, that play an important role in osteoclast fusion by a RANKL‐independent mechanism. J. Cell. Biochem. 106: 563–569, 2009. © 2009 Wiley‐Liss, Inc.     

Targeting bone remodeling by isoflavone and 3,3'-diindolylmethane in the context of prostate cancer bone metastasis

Prostate cancer (PCa) bone metastases have long been believed to be osteoblastic because of bone remodeling leading to the formation of new bone. However, recent studies have shown increased osteolytic activity in the beginning stages of PCa bone metastases, suggesting that targeting both osteolytic and osteoblastic mediators would likely inhibit bone remodeling and PCa bone metastasis. In this study, we found that PCa cells could stimulate differentiation of osteoclasts and osteoblasts through the up-regulation of RANKL, RUNX2 and osteopontin, promoting bone remodeling. Interestingly, we found that formulated isoflavone and 3,3'-diindolylmethane (BR-DIM) were able to inhibit the differentiation of osteoclasts and osteoblasts through the inhibition of cell signal transduction in RANKL, osteoblastic, and PCa cell signaling. Moreover, we found that isoflavone and BR-DIM down-regulated the expression of miR-92a, which is known to be associated with RANKL signaling, EMT and cancer progression. By pathway and network analysis, we also observed the regulatory effects of isoflavone and BR-DIM on multiple signaling pathways such as AR/PSA, NKX3-1/Akt/p27, MITF, etc. Therefore, isoflavone and BR-DIM with their multi-targeted effects could be useful for the prevention of PCa progression, especially by attenuating bone metastasis mechanisms.     

Hippuric acid and 3-(3-hydroxyphenyl) propionic acid inhibit murine osteoclastogenesis through RANKL-RANK independent pathway

Nutritional factors influence bone development. Previous studies demonstrated that bone mass significantly increased with suppressed bone resorption in early life of rats fed with AIN-93G semi-purified diets supplemented with 10% whole blueberry (BB) powder for 2 weeks. However, the effects of increased phenolic acids in animal serum due to this diet on bone and bone resorption were unclear. This in vitro and in ex vivo study examined the effects of phenolic hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA) on osteoclastic cell differentiation and bone resorption. We cultured murine osteoclast (macrophage) cell line, RAW 264.7 cells, and hematopoietic osteoclast progenitor cells (isolated from 4-week-old C57BL6/J mice) with 50 ng/ml of receptor activator of nuclear factor κ-Β ligand (RANKL). Morphologic studies showed decreased osteoclast number with treatment of 2.5% mouse serum from BB diet–fed animals compared with those treated with serum from standard casein diet–fed mice in both RAW 264.7 cell and primary cell cultures. HA and 3-3-PPA, but not 3–4-PPA, had dose-dependent suppressive effects on osteoclastogenesis and osteoclast resorptive activity in Corning osteo-assay plates. Signaling pathway analysis showed that after pretreatment with HA or 3-3-PPA, RANKL-stimulated increase of osteoclastogenic markers, such as nuclear factor of activated T-cells, cytoplasmic 1 and matrix metallopeptidase 9 gene/protein expression were blunted. Inhibitory effects of HA and 3-3-PPA on osteoclastogenesis utilized RANKL/RANK independent mediators. The study revealed that HA and 3-3-PPA significantly inhibited osteoclastogenesis and bone osteoclastic resorptive activity.     

Direct effects of osteoprotegerin on human bone cell metabolism

Purpose

Osteoprotegerin (OPG) affects bone metabolism by intercepting the RANK-RANKL interaction which prevents osteoclastic differentiation and consequently reduces bone resorption. Different bone phenotypes of mice overexpressing OPG and of mice with knockdown of receptor activator of NF-κB (RANK) or RANK-ligand (RANKL) suggest that the mechanism of action of the OPG-RANKL-RANK system in regulating bone remodeling is not completely understood. Furthermore, OPG increases bone mass and density independently from reduced osteoclastogenesis which is consistent with the possibility that OPG may directly affect bone metabolism beyond its known role as decoy receptor for RANKL.

Methods

We treated primary human osteoblastic cells with OPG and inhibitory anti-RANKL antibodies and measured cellular ALP activity, in vitro mineralization, vitronectin receptor protein expression and ERK phosphorylation. We also analyzed the mRNA co-expression of ALP and OPG ex vivo in bone biopsies from acute and old stable vertebral fractures.

Results

OPG directly increased ALP activity and in vitro mineralization of HOC, enhanced expression of the vitronectin receptor thereby increasing adherence of HOC to vitronectin and stimulated ERK phosphorylation. All OPG-mediated effects could be prevented by RANKL antibodies or RANKL-siRNA transfection and MAPK inhibitor PD98059 reduced the stimulatory effect of OPG on integrin αv expression. In acutely fractured vertebrae OPG and ALP mRNA expression was significantly increased compared to stable vertebral fractures. In conclusion, OPG exerts direct osteoanabolic effects on HOC metabolism via RANKL in addition to its well described role as decoy receptor for RANKL.     

Functions of RANKL/RANK/OPG in bone modeling and remodeling

The discovery of the RANKL/RANK/OPG system in the mid 1990s for the regulation of bone resorption has led to major advances in our understanding of how bone modeling and remodeling are regulated. It had been known for many years before this discovery that osteoblastic stromal cells regulated osteoclast formation, but it had not been anticipated that they would do this through expression of members of the TNF superfamily: receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG), or that these cytokines and signaling through receptor activator of NF-κB (RANK) would have extensive functions beyond regulation of bone remodeling. RANKL/RANK signaling regulates osteoclast formation, activation and survival in normal bone modeling and remodeling and in a variety of pathologic conditions characterized by increased bone turnover. OPG protects bone from excessive resorption by binding to RANKL and preventing it from binding to RANK. Thus, the relative concentration of RANKL and OPG in bone is a major determinant of bone mass and strength. Here, we review our current understanding of the role of the RANKL/RANK/OPG system in bone modeling and remodeling.     

Inhibition of osteoblastic metalloproteinases in mice prevents bone loss induced by oestrogen deficiency

Matrix metalloproteinases (MMPs) are key mediators in extra-cellular matrix remodelling and implicated primarily in bone growth, and particularly in osteoclastic bone resorption. We hypothesise that MMPs have a role in the increased bone remodelling resulting from oestrogen deficiency. Transgenic (TG) mice overexpressing TIMP-1 in their osteoblastic cells and their wild-type (WT) littermates were ovariectomised. One month after surgery, bone mineral density (BMD) and bone microarchitecture were assessed. Primary cells from WT and TG mice were used to determine how TIMP-1 affects osteoclast and osteoblastic cells. The reduction of BMD induced by ovariectomy in WT mice was not observed in the transgenic mice. The transgene overexpression also dampened the post-ovariectomy increase in bone resorption in contrast to the WT mice. In vivo, osteoclastic surfaces and D-pyridinoline were not increased in TG mice, and ex vivo, the differentiation of osteoclasts from TG bone marrow precursor cells were unaffected by in vivo oestrogen deficiency or treatment. We showed also that TIMP-1 overexpression reduces and delays the osteoblastic proliferation and differentiation respectively, and reduced the generation of the active form of TGFbeta1 in the supernatant of TG osteoblasts. Our findings support the hypothesis that in vivo inhibition of osteoblastic MMPs prevented the bone loss induced by oestrogen deficiency, with a significant decrease in bone resorption. This effect was presumably resulting from (1) a direct inhibition of osteoclastic resorption activity by the TIMP-1 and (2) the modification in the local activation of extra-cellular signalling factors such as TGFbeta1 and the OPG/RANKL ratio.     

OPG/RANKL/RANK系统与骨破坏性疾病

近年来发现的OPG/RANKL/RANK系统在破骨细胞生成中起着至关重要的作用,是骨骼生理研究领域的重大进展。成骨细胞、骨髓基质细胞、激活的T淋巴细胞表达RANKL,与破骨细胞前体细胞或成熟破骨细胞表面上的RANK结合后,促进破骨细胞的分化及骨吸收活性。成骨细胞及骨髓基质细胞分泌表达OPG可与RANKL竞争性结合,从而阻断RANKL与RANK之间的相互作用。体内多种激素或因子通过影响骨髓微环境内的OPG/RANKL比率来调节骨代谢。此外,乳腺上皮细胞表达有RANK,孕期在性激素的诱导下可表达RANKL,OPG/RANKL/RANK系统在孕期乳腺发育以及母体向胎儿的钙转运过程中发挥重要作用。阻断RANKL/RANK通路有望给骨质疏松、类风湿关节炎及癌症骨转移等骨破坏性疾病的治疗开辟新的途径。进一步研究应了解OPG/RANKL/RANK系统与其它信号传导途径的关系,重视骨骼、免疫及内分泌系统之间的相互作用。目前,开发与OPG功能相似或促进其表达的合成药物有可能成为具有良好经济效益和社会效益的产业。     

Dexamethasone inhibits bone resorption by indirectly inducing apoptosis of the bone-resorbing osteoclasts via the action of osteoblastic cells

Although glucocorticoids (GCs) are physiologically essentialfor bone metabolism, it is generally accepted that high dosesof GCs cause bone loss through a combination of decreased boneformation and increased bone resorption. However, the actionof GCs on mature osteoclasts remains contradictory. In thisstudy, we have examined the effect of GCs on osteoclasticbone-resorbing activity and osteoclast apoptosis, by using twodifferent cell types, rabbit unfractionated bone cells andhighly enriched mature osteoclasts (>95% of purity).Dexamethasone (Dex, 10^-10–10^-7 M) inhibited resorption pit formation on a dentine slice by the unfractionated bone cells in a dose- and time-dependent manner.However, Dex had no effect on the bone-resorbing activity of the isolated mature osteoclasts. When the isolated osteoclastswere co-cultured with rabbit osteoblastic cells, the osteoclastic bone resorption decreased in response to Dex,dependent on the number of osteoblastic cells. Like the effecton the bone resorption, Dex induced osteoclast apoptosis in cultures of the unfractionated bone cells, whereas it did not promote the apoptosis of the isolated osteoclasts. An inhibitorof caspases, Z-Asp-CH2-DCB attenuated both the inhibitory effecton osteoclastic bone resorption and the stimulatory effect onthe osteoclast apoptosis. In addition, the osteoblastic cellswere required for the osteoclast apoptosis induced by Dex. These findings indicate that the main target cells of GCs arenon-osteoclastic cells such as osteoblasts and that GCsindirectly inhibit bone resorption by inducing apoptosis ofthe mature osteoclasts through the action of non-osteoclasticcells. This study expands our knowledge about the multifunctional roles of GCs in bone metabolism.     

Osteoclast‐specific Dicer gene deficiency suppresses osteoclastic bone resorption

Osteoclasts are unique cells that resorb bone, and are involved in not only bone remodeling but also pathological bone loss such as osteoporosis and rheumatoid arthritis. The regulation of osteoclasts is based on a number of molecules but full details of these molecules have not yet been understood. MicroRNAs are produced by Dicer cleavage an emerging regulatory system for cell and tissue function. Here, we examine the effects of Dicer deficiency in osteoclasts on osteoclastic activity and bone mass in vivo. We specifically knocked out Dicer in osteoclasts by crossing Dicer flox mice with cathepsin K‐Cre knock‐in mice. Dicer deficiency in osteoclasts decreased the number of osteoclasts (N.Oc/BS) and osteoclast surface (Oc.S/BS) in vivo. Intrinsically, Dicer deficiency in osteoclasts suppressed the levels of TRAP positive multinucleated cell development in culture and also reduced NFATc1 and TRAP gene expression. MicroRNA analysis indicated that expression of miR‐155 was suppressed by RANKL treatment in Dicer deficient cells. Dicer deficiency in osteoclasts suppressed osteoblastic activity in vivo including mineral apposition rate (MAR) and bone formation rate (BFR) and also suppressed expression of genes encoding type I collagen, osteocalcin, Runx2, and Efnb2 in vivo. Dicer deficiency in osteoclasts increased the levels of bone mass indicating that the Dicer deficiency‐induced osteoclastic suppression was dominant over Dicer deficiency‐induced osteoblastic suppression. On the other hand, conditional Dicer deletion in osteoblasts by using 2.3 kb type I collagen‐Cre did not affect bone mass. These results indicate that Dicer in osteoclasts controls activity of bone resorption in vivo. J. Cell. Biochem. 109: 866–875, 2010. © 2009 Wiley‐Liss, Inc.     

T Cells?Induce Pre-Metastatic Osteolytic?Disease and Help Bone Metastases Establishment in a Mouse Model of Metastatic Breast Cancer

Bone metastases, present in 70% of patients with metastatic breast cancer, lead to skeletal disease, fractures and intense pain, which are all believed to be mediated by tumor cells. Engraftment of tumor cells is supposed to be preceded by changes in the target tissue to create a permissive microenvironment, the pre-metastatic niche, for the establishment of the metastatic foci. In bone metastatic niche, metastatic cells stimulate bone consumption resulting in the release of growth factors that feed the tumor, establishing a vicious cycle between the bone remodeling system and the tumor itself. Yet, how the pre-metastatic niches arise in the bone tissue remains unclear. Here we show that tumor-specific T cells induce osteolytic bone disease before bone colonization. T cells pro-metastatic activity correlate with a pro-osteoclastogenic cytokine profile, including RANKL, a master regulator of osteoclastogenesis. In vivo inhibition of RANKL from tumor-specific T cells completely blocks bone loss and metastasis. Our results unveil an unexpected role for RANKL-derived from T cells in setting the pre-metastatic niche and promoting tumor spread. We believe this information can bring new possibilities for the development of prognostic and therapeutic tools based on modulation of T cell activity for prevention and treatment of bone metastasis.     

Osteoblastic regulation of osteoclast survival: effect of calcitriol

Throughout life, bone is remodelled in a dynamic process which results in a balance between bone formation by osteoblasts and bone resorption by osteoclasts. It is now clearly established that osteoblasts/stromal cells are crucial for differentiation of osteoclasts, through a mechanism involving cell-to-cell contact. However, the possible involvement of osteoblasts and stromal cells in the survival of osteoclasts has not yet been clearly demonstrated. In this study, we assessed the influence of cellular microenvironment, especially osteoblasts, on the osteoclast survival. Our results have shown significant differences in osteoclastic survival between unfractionated bone cells and pure osteoclasts. Furthermore, we have shown that addition of 1.25(OH)2D3 to unfractionated bone cells resulted in a dose-dependent increase in osteoclast survival. Finally, we have shown that a conditioned medium obtained from rat osteoblastic cells cultured with calcitriol was able to increase significantly survival of pure osteoclasts. Taken together, these results strongly suggest that osteoblastic cells present in the bone microenvironment might play a role in the osteoclastic survival by producing soluble factor which modulate osteoclast apoptosis.     

Clinical development of anti-RANKL therapy

The receptor activator of nuclear factor-kappaB ligand (RANKL), its cognate receptor RANK, and its natural decoy receptor osteoprotegerin have been identified as the final effector molecules of osteoclastic bone resorption. This has provided an ideal target for therapeutic interventions in metabolic bone disease. As described in previous reviews in this supplement, RANKL signaling is required for osteoclast differentiation, activation, and survival. Furthermore, in vivo inhibition of RANKL leads to immediate osteoclast apoptosis, and there are no in vivo models of bone resorption that are refractory to RANKL inhibition. Thus, the only step remaining in the development of a clinical intervention is the generation of a safe, effective, and specific drug that can inhibit RANKL in humans. Here we review the clinical development of denosumab (formerly known as AMG 162), which is a fully human mAb directed against RANKL. This discussion includes the breadth of 21 human studies that have led to the current phase 3 clinical trials seeking approval for use of this agent to treat postmenopausal women with low bone mineral density (osteoporosis) and patients with metastatic lytic bone lesions (multiple myeloma, and prostate and breast cancer).     

Osteoblastic cells induce fusion and activation of osteoclasts through a mechanism independent of macrophage-colony-stimulating factor production

Fusion and activation of osteoclasts are the final two events in osteoclastic bone resorption. To investigate the regulatory mechanism of these events, mononuclear osteoclasts (preosteoclasts, pOCs) were isolated from co-cultures of mouse osteoblastic cells and bone marrow cells. Most of the pOCs cultured without any additives died within 12 h. Survival of pOCs was supported by addition of either osteoblastic cells or macrophage-colony-stimulating factor (M-CSF). pOCs began to fuse with each other after culture for 12 h in the presence of osteoblastic cells or M-CSF. However, the properties of multinucleated osteoclast-like cells (OCLs) induced by osteoblastic cells were considerably different from those induced by M-CSF. Fusion of pOCs induced by osteoblastic cells was retarded after culture for 24 h. In contrast, M-CSF-induced fusion of pOCs continued throughout the 48-h culture period, which was not inhibited by addition of calcitonin. When pOCs together with osteoblastic cells were cultured for 48 h on dentine slices, many resorption pits were formed on the slices. Calcitonin completely inhibited the fusion and pit-forming activity of pOCs treated with osteoblastic cells. Resorption pits were hardly detected on dentine slices in pOC cultures treated with M-CSF. Osteoblastic cells prepared from osteopetrotic (op/op) mice, which cannot produce functional M-CSF, stimulated the fusion and pit-forming activity of pOCs. Recombinant RANKL (receptor activator of NF-kappaB ligand), a cytokine which is produced by osteoblastic cells and is responsible for osteoclast differentiation, induced the fusion and pit-forming activity of pOCs. These results suggested that osteoblastic cells are involved in fusion and activation of osteoclasts through a mechanism independent of M-CSF production. RANKL appears to be responsible for fusion and activation of osteoclasts induced by osteoblastic cells.     

The OPG/RANKL/RANK system in metabolic bone diseases

The OPG/RANKL/RANK cytokine system is essential for osteoclast biology. Various studies suggest that human metabolic bone diseases are related to alterations of this system. Here we summarize OPG/RANKL/RANK abnormalities in different forms of osteoporoses and hyperparathyroidism. Skeletal estrogen agonists (including 17beta-estradiol, raloxifene, and genistein) induce osteoblastic OPG production through estrogen receptor-alpha activation in vitro, while immune cells appear to over-express RANKL in estrogen deficiency in vivo. Of note, OPG administration can prevent bone loss associated with estrogen deficiency as observed in both animal models and a small clinical study. Glucocorticoids and immunosuppressants concurrently up-regulate RANKL and suppress OPG in osteoblastic cells in vitro, and glucocorticoids are among the most powerful drugs to suppress OPG serum levels in vivo. As for mechanisms of immobilization-induced bone loss, it appears that mechanical strain inhibits RANKL production through the ERK 1/2 MAP kinase pathway and up-regulates OPG production in vitro. Hence, lack of mechanical strainduring immobilization may favor an enhanced RANKL-to-OPG ratio leading to increased bone loss. As for hyperparathyroidism, chronic PTH exposure concurrently enhances RANKL production and suppresses OPG secretion through activation of osteoblastic protein kinase A in vitro which would favour increased osteoclastic activity. In sum, the capacity for OPG to antagonize the increases in bone loss seen in many rodent models of metabolic bone disease implicates RANKL/OPG imbalances as the likely etiology and supports the potential role for a RANKL antagonist as a therapeutic intervention in these settings.