Giant Osteoclasts Research Articles

Effects of androgen deprivation therapy and bisphosphonate treatment on bone in patients with metastatic castration-resistant prostate cancer: Results from the University of Washington Rapid Autopsy Series

Qualitative and quantitative bone features were determined in nondecalcified and decalcified bone from 20 predetermined bone sites in each of 44 patients who died with castration-resistant prostate cancer (CRPC), some of which received bisphosphonate treatment (BP) in addition to androgen-deprivation therapy (ADT). Thirty-nine of the 44 patients (89%) had evidence of bone metastases. By histomorphometric analysis, these bone metastases were associated with a range of bone responses from osteoblastic to osteolytic with a wide spectrum of bone responses often seen within an individual patient. Overall, the average bone volume/tissue volume (BV/TV) was 25.7%, confirming the characteristic association of an osteoblastic response to prostate cancer bone metastasis when compared with the normal age-matched weighted mean BV/TV of 14.7%. The observed new bone formation was essentially woven bone, and this was a localized event. In comparing BV/TV at metastatic sites between patients who had received BP treatment and those who had not, there was a significant difference (28.6% versus 19.3%, respectively). At bone sites that were not invaded by tumor, the average BV/TV was 10.1%, indicating significant bone loss owing to ADT that was not improved (11%) in those patients who had received BPs. Surprisingly, there was no significant difference in the number of osteoclasts present at the metastatic sites between patients treated or not treated with BPs, but in bone sites where the patient had been treated with BPs, giant osteoclasts were observed. Overall, 873 paraffin-embedded specimens and 661 methylmethacrylate-embedded specimens were analyzed. Our results indicate that in CRPC patients, ADT induces serious bone loss even in patients treated with BP. Furthermore, in this cohort of patients, BP treatment increased BV and did not decrease the number of osteoclasts in prostate cancer bone metastases compared with bone metastases from patients who did not receive BP.

Rapamycin inhibits osteoclast formation in giant cell tumor of bone through the C/EBPβ - MafB axis

Giant cell tumor (GCT) of bone is a benign type of tumor, but the presence of hyperactive multinucleated giant osteoclasts cause local osteolytic lesions, increasing morbidity in patients. To specifically target hyperactive multinucleated giant osteoclasts in GCTs, one would envisage the usage of osteoclast inhibitors or genetic modulation of osteoclastogenesis. Recently, we have found that the translationally regulated balance between the transcription factor C/EBPβ long (LAP) and short (LIP) protein isoforms regulates osteoclast differentiation. Here, we report that GCTs express high levels of the LIP C/EBPβ isoform, which in mice cause giant osteoclast formation. In mice, inhibition of mTOR activity by rapamycin decreased osteoclast differentiation by shifting the alternative translation initiation of C/EBPβ isoforms towards LAP. Similarly, rapamycin treatment of GCT cell cultures derived from seven different patients strongly reduced formation of giant osteoclasts and bone resorption. This was accompanied by an increase in MafB, previously shown to be the mediator of the effect of rapamycin on osteoclast differentiation in mice. These data suggest that C/EBPβ is a determinant of giant osteoclast formation in GCT and that pharmacological adjustment of the C/EBPβ isoform ratio could serve as a potential novel therapeutic approach.

NF-κB Signaling Participates in Both RANKL- and IL-4–Induced Macrophage Fusion: Receptor Cross-Talk Leads to Alterations in NF-κB Pathways

NF-κB activation is essential for receptor activator for NF-κB ligand (RANKL)-induced osteoclast formation. IL-4 is known to inhibit the RANKL-induced osteoclast differentiation while at the same time promoting macrophage fusion to form multinucleated giant cells (MNG). Several groups have proposed that IL-4 inhibition of osteoclastogenesis is mediated by suppressing the RANKL-induced activation of NF-κB. However, we found that IL-4 did not block proximal, canonical NF-κB signaling. Instead, we found that IL-4 inhibited alternative NF-κB signaling and induced p105/50 expression. Interestingly, in nfκb1(-/-) bone marrow-derived macrophages (BMM), the formation of both multinucleated osteoclast and MNG induced by RANKL or IL-4, respectively, was impaired. This suggests that NF-κB signaling also plays an important role in IL-4-induced macrophage fusion. Indeed, we found that the RANKL-induced and IL-4-induced macrophage fusion were both inhibited by the NF-κB inhibitors IκB kinase 2 inhibitor and NF-κB essential modulator inhibitory peptide. Furthermore, overexpression of p50, p65, p52, and RelB individually in nfκb1(-/-) or nfκb1(+/+) BMM enhanced both giant osteoclast and MNG formation. Interestingly, knockdown of nfκb2 in wild-type BMM dramatically enhanced both osteoclast and MNG formation. In addition, both RANKL- and IL-4-induced macrophage fusion were impaired in NF-κB-inducing kinase(-/-) BMM. These results suggest IL-4 influences NF-κB pathways by increasing p105/p50 and suppressing RANKL-induced p52 translocation and that NF-κB pathways participate in both RANKL- and IL-4-induced giant cell formation.

Dynamics of giant osteoclast formation in bisphosphonate-treated animals

Dynamics of giant osteoclast formation in bisphosphonate-treated animals

Impaired mesenchymal stem cell differentiation and osteoclastogenesis in mice deficient for Igf2-P2 transcripts

During embryonic development, Igf2 gene transcription is highly regulated through the use of several promoters whose specific roles are not defined. Here, we show that loss-of-function of one of these promoters, Igf2-P2, results in growth defects that are temporally and quantitatively different from those seen in Igf2-null mutants. In particular, Igf2-P2 mutants exhibit skeletal abnormalities characterized by thin and short bones with reduced mineralization and medullar cavity and with altered bone remodeling. These abnormalities are associated with decreased numbers of embryonic mesenchymal chondroprogenitors, adult mesenchymal stem cells and osteoprogenitors. Differentiation of osteoprogenitors into osteoblasts is impaired in the Igf2-P2 mutant mice in a cell-autonomous manner, and osteopontin is a target of the IGF2 signaling pathway during this differentiation. Igf2-P2 mutant mice also display impaired formation of giant osteoclasts owing to a defective micro-environment. These results support a model wherein transcriptional activity of the Igf2-P2 promoter regulates the fate of mesenchymal progenitors during bone development and remodeling in the adult, and regulates osteogenesis in a cell-autonomous and non-autonomous manner.

Osteoclastic Metabolism of 25(OH)-Vitamin D3: A Potential Mechanism for Optimization of Bone Resorption

The extrarenal synthesis of 1α,25 dihydroxyvitamin D3 (1,25D) has been demonstrated in a number of cell types including osteoblasts and cells of the monocyte/macrophage lineage. The skeleton appears responsive to serum levels of the 1,25D precursor, 25 hydroxyvitamin D3 (25D), in terms of bone mineralization parameters. The effect of metabolism of 25D into active 1,25D by osteoclast lineage cells is unknown. We found that CYP27B1 mRNA expression increased with exposure of human peripheral blood mononuclear cells (PBMCs) to macrophage colony-stimulating factor in the presence or absence of receptor activator of nuclear factor-κB ligand. Consistent with this, human osteoclast cultures incubated with 25D produced measurable quantities of 1,25D. Osteoclast formation from either mouse RAW264.7 cells or human PBMCs in the presence of physiological concentrations of 25D resulted in significant up-regulation of the key osteoclast transcription factor, nuclear factor of activated T cells-c1 in PBMCs and a number of key osteoclast marker genes in both models. The expression of the osteoblast coupling factor, ephrin-b2, was also increased in the presence of 25D. Levels of CYP27B1 and nuclear factor of activated T cells-1 mRNA correlated during osteoclastogenesis and also in a cohort of human bone samples. CYP27B1 short-hairpin RNA knockdown in RAW264.7 cells decreased their osteoclastogenic potential. 25D dose dependently reduced the resorptive capacity of PBMC-derived osteoclasts without compromising cell viability. 25D also reduced resorption by RAW264.7- and giant cell tumor-derived osteoclasts. Conversely, osteoclasts formed from vitamin D receptor-null mouse splenocytes had increased resorptive activity compared with wild-type cells. We conclude that 25D metabolism is an important intrinsic mechanism for optimizing osteoclast differentiation, ameliorating osteoclast activity, and potentially promoting the coupling of bone resorption to formation.

Severe developmental bone phenotype in ClC-7 deficient mice

Bone development is dependent on the functionality of three essential cell types: chondrocytes, osteoclasts and osteoblasts. If any of these cell types is dysfunctional, a developmental bone phenotype can result. The bone disease osteopetrosis is caused by osteoclast dysfunction or impaired osteoclastogenesis, leading to increased bone mass. In ClC-7 deficient mice, which display severe osteopetrosis, the osteoclast malfunction is due to abrogated acidification of the resorption lacuna. This study sought to investigate the consequences of osteoclast malfunction on bone development, bone structure and bone modeling/remodeling in ClC-7 deficient mice. Bones from wildtype, heterozygous and ClC-7 deficient mice were examined by bone histomorphometry and immunohistochemistry. ClC-7 deficient mice were found to have a severe developmental bone phenotype, characterized by dramatically increased bone mass, a high content of cartilage remnants, impaired longitudinal and radial growth, as well as lack of compact cortical bone development. Indices of bone formation were reduced in ClC-7 deficient mice; however, calcein labeling indicated that mineralization occurred on most trabecular bone surfaces. Osteoid deposition had great regional variance, but an osteopetrorickets phenotype, as observed in oc/oc mice, was not apparent in the ClC-7 deficient mice. A striking finding was the presence of very large abnormal osteoclasts, which filled the bone marrow space within the ClC-7 deficient bones. The development of these giant osteoclasts could be due to altered cell fate of the ClC-7 deficient osteoclasts, caused by increased cellular fusion and/or prolonged osteoclast survival. In summary, malfunctional ClC-7 deficient osteoclasts led to a severe developmental bone phenotype including abnormally large and non-functional osteoclasts. Bone formation paremeters were reduced; however, bone formation and mineralization were found to be heterogenous and continuing.

Thalidomide dramatically improves the symptoms of early‐onset Sarcoidosis/Blau syndrome: Its possible action and mechanism

Early-onset sarcoidosis (EOS), which occurs in children younger than 5 years of age, is associated with granulomatous lesions and a sporadic genetic mutation of the nucleotide-binding oligomerization domain 2 that causes constitutive NF-kappaB activation. The symptoms of EOS can be uncontrollable, progressive, and associated with profound complications. However, appropriate therapy is still under investigation. The aim of this study was to assess the efficacy of thalidomide in patients with severe EOS, based on etiology supporting an initial role of NF-kappaB in activation of this disease. Thalidomide was given to 2 patients with EOS (a 16-year-old girl and an 8-year-old boy) at an initial dosage of 2 mg/kg/day, and the dosage was increased if necessary. To elucidate the mechanism of the drug, peripheral blood monocytes were isolated from the patients and stimulated with cytokines (macrophage colony-stimulating factor, tumor necrosis factor alpha, and interleukin-4), and their ability to form multinucleated giant cells (MGCs) and osteoclasts was measured. Both patients showed dramatic improvement of their clinical symptoms (alleviation of fever and optic nerve papillitis, achievement of a response according to the American College of Rheumatology Pediatric 50 and Pediatric 70 criteria) and laboratory findings. Monocytes from patients with EOS had a greater ability to survive and induce MGCs and osteoclasts than those from healthy control subjects. The formation of MGCs and osteoclasts was inhibited by the presence of thalidomide. The ability of thalidomide to improve clinical symptoms and laboratory findings in patients with EOS indicates a central role for NF-kappaB activity in this disorder. Inhibition of IKK might be a pharmacologic action by which thalidomide down-regulates NF-kappaB signaling. Thalidomide may be an effective medication in patients with severe complications of EOS, including ocular involvement.

Vitamin D and the Parenteral Nutrition Patient

Vitamin D is a prohormone produced in the skin epidermis when irradiated with sunlight or ultraviolet light B. It is also absorbed from food or supplements. Vitamin D must be converted to 25-hydroxyvitamin D3 (circulating form) and finally to the hormone, 1a,25-dihydroxyvitamin D3, before it can function. This hormone acts through a single nuclear receptor. The details of these conversions and molecular biology of the action of vitamin D3 are summarized. The physiologic functions of vitamin D have been expanded beyond the mineralization of the skeleton to include modulation of the immune system, terminal differentiation in several tissues, suppression of malignant cells, and anabolic activity in the skeleton and in the renal-cardiovascular system. Epidemiologic studies have associated vitamin D deficiency with an increased risk of colorectal and breast cancers and an increased risk of autoimmune diseases, such as multiple sclerosis, type 1 diabetes, and cardiovascular events. Thus, vitamin D is essential not only for the skeleton but also many other organ systems. Recommendations for 25-hydroxyvitamin D3 levels for PN patients are presented.

Whither goest the lifeless osteoclast?

Evaluation of: Weinstein RS, Roberson PK, Manolagas SC: Giant osteoclast formation and long-term oral bisphosphonate therapy. N. Engl. J. Med. 360, 53–62 (2009). Giant osteoclasts are pathognomonic in Paget’s Disease, secondary hyperparathyroidism, giant cell tumors and fibrous dysplasia. Weinstein et al. provided a detailed histological analysis of abnormal hypernucleated osteoclasts in half of a set of biopsies from patients who received clinical doses of alendronate, a bisphosphonate inhibitor of bone resorption. In addition, 30% of the osteoclasts were apoptotic, a feature not found in the other clinical conditions. Their report reveals new information about skeletal effects of alendronate in addition to known effects to inhibit osteoclastic bone resorption. Their report raises new questions about the lifecycle of the human osteoclast, effects of bisphosphonates on cell fusion and on efferocytosis (removal of apoptotic cells), significance, if any, of the abnormal osteoclasts, and distinguishing featu...

Bone formation on rough, but not polished, subcutaneously implanted Ti surfaces is preceded by macrophage accumulation

Implanted rough surfaces have long been associated with the accumulation of macrophages and other cells of the monocytic lineage such as foreign body giant cells and osteoclasts. As cells of the moncytic lineage are part of the immune system, the response of this cell family to biomaterials has attracted wide concern. This study compared events at the interface of implant surface topographies with varied roughness in a rat subcutaneous model. Titanium-coated epoxy replicas of machined, etched, blasted, titanium-plasma-sprayed (TPS), sandblasted-and-etched (SLA), micromachined, and polished surfaces were implanted for up to 11 weeks, and processed for light or electron microscopy or immunohistochemistry for ED1, a marker for recruited macrophages. Initially, healing appeared similar among all surfaces, the frequency of mineralization followed the order of SLA, micromachined, TPS, machined, etched, blasted, and polished surfaces. On the SLA surface macrophages, as identified by both ultrastructural morphology and immunohistochemistry were the predominant cell type at 1 week and persisted until mineralization occurred as early as 2 weeks. On smoother surfaces collagenous matrix predominated at 2 weeks and subsequently increased with time. There, thus, appears to be two routes to bone-like tissue formation on Ti implants in this rat subcutaneous model; macrophage-mediated and macrophage-independent dense collagenous-matrix-associated.

Giant osteoclasts after long-term bisphosphonate therapy: diagnostic challenges

A 55-year-old woman with a 5-year history of osteoporosis treated for 4 years with an oral aminobisphosphonate presented with a recent vertebral fracture. A bone biopsy specimen revealed giant osteoclasts with more than 40 nuclear profiles. Bone mineral density determinations, measurement of serum 25-hydroxyvitamin D, intact parathyroid hormone, calcium, inorganic phosphorus, alkaline phosphatase and creatinine levels, urinary excretion levels of the N-telopeptide of type 1 collagen, and bone biopsy. Examination of the patient and review of the bone specimen. Giant osteoclasts after long-term bisphosphonate use, without evidence of malignancy. Interpretation of the bone biopsy specimen listed several bone disorders. The bone specimen was reviewed and the histological differential diagnosis was carefully considered. The giant osteoclasts were detached from bone and frequently apoptotic in a normal marrow stroma, with low-to-normal amounts of osteoid and osteoblasts. These features are typical of giant osteoclast formation after long-term aminobisphosphonate therapy. the patient was reassured that the bone findings were unlikely to be detrimental. Aminobisphosphonate treatment was reinstituted, and 1 year later the patient was asymptomatic.

Giant osteoclasts detected in bone biopsy samples after alendronate therapy

Giant osteoclasts detected in bone biopsy samples after alendronate therapy

Giant Osteoclast Formation and Long-Term Oral Bisphosphonate Therapy

Giant Osteoclast Formation and Long-Term Oral Bisphosphonate Therapy

Should we be concerned about giant osteoclasts?

Should we be concerned about giant osteoclasts?

Oral bisphosphonates related to giant osteoclast formation?

Oral bisphosphonates related to giant osteoclast formation?

Giant Osteoclast Formation and Long-Term Oral Bisphosphonate Therapy

Bisphosphonates decrease bone resorption and are commonly used to treat or prevent osteoporosis. However, the effect of bisphosphonates on their target cells remains enigmatic, since in patients benefiting from therapy, little change, if any, has been observed in the number of osteoclasts, which are the cells responsible for bone resorption. We examined 51 bone-biopsy specimens obtained after a 3-year, double-blind, randomized, placebo-controlled, dose-ranging trial of oral alendronate to prevent bone resorption among healthy postmenopausal women 40 through 59 years of age. The patients were assigned to one of five groups: those receiving placebo for 3 years; alendronate at a dose of 1, 5, or 10 mg per day for 3 years; or alendronate at a dose of 20 mg per day for 2 years, followed by placebo for 1 year. Formalin-fixed, undecalcified planar sections were assessed by bone histomorphometric methods. The number of osteoclasts was increased by a factor of 2.6 in patients receiving 10 mg of alendronate per day for 3 years as compared with the placebo group (P<0.01). Moreover, the number of osteoclasts increased as the cumulative dose of the drug increased (r=0.50, P<0.001). Twenty-seven percent of these osteoclasts were giant cells with pyknotic nuclei that were adjacent to superficial resorption cavities. Furthermore, giant, hypernucleated, detached osteoclasts with 20 to 40 nuclei were found after alendronate treatment had been discontinued for 1 year. Of these large cells, 20 to 37% were apoptotic, according to both their morphologic features and positive findings from in situ end labeling. Long-term alendronate treatment is associated with an increase in the number of osteoclasts, which include distinctive giant, hypernucleated, detached osteoclasts that are undergoing protracted apoptosis.

Giant Osteoclast Formation and Long-Term Oral Bisphosphonate Therapy

Giant Osteoclast Formation and Long-Term Oral Bisphosphonate Therapy

IGF2 modulates the microenvironment for osteoclastogenesis

We previously reported that hypoxic stress enhanced osteoclast differentiation via increasing insulin-like growth factor 2 (IGF2) production. However, the mechanisms underlying IGF2 stimulation remains unknown. In this study, we investigated the molecular mechanisms of osteoclastogenesis by IGF2 treatment. Primary mouse bone marrow cells were cultured with IGF2. Total RNAs were applied to a DNA microarray analysis, and quantitative RT-PCR was then used to confirm the microarray data and clarify which cells expressed the relative genes. The most interesting findings were the upregulations of CXC chemokine ligand 7 (CXCL7) expression in stromal cells and stromal cell-derived factor 1 (SDF1) expression in osteoblastic cells with IGF2 treatment. The addition of exogenous SDF1 to CXCL7 increased the number of osteoclasts and promoted the formation of giant osteoclasts. These results suggest that IGF2 modulates the microenvironment around osteoclast precursor cells. SDF1 together with CXCL7 may promote the formation of giant osteoclasts.

Gap junctional communication in human osteoclasts in vitro and in vivo

Bone-forming cells are known to be coupled by gap junctions, formed primarily by connexin43 (Cx43). The role of Cx43 in osteoclasts has so far only been studied in rodents, where Cx43 is important for fusion of mononuclear precursors to osteoclasts. Given the potential importance for human diseases with pathologically altered osteoclasts, we asked whether a similar influence of Cx43 can also be observed in osteoclasts of human origin. For this purpose, Cx43 mRNA expression was studied in a time course experiment of human osteoclast differentiation by RT-PCR. Localization of Cx43 in these cells was determined by immunohistochemistry and confocal microscopy. For the assessment of the effect of gap junction inhibition on cell fusion, gap junctions were blocked with heptanol during differentiation of the cells and the cells were then evaluated for multinuclearity. Paraffin sections of healthy bone and bone from patients with Paget's disease and giant cell tumour of the bone were used to study Cx43 expression in vivo. We found mRNA and protein expression of Cx43 in fully differentiated osteoclasts as well as in precursor cells. This expression decreased in the course of differentiation. Consistently, we found a lower expression of Cx43 in osteoclasts than in bone marrow precursor cells in the histology of healthy human bone. Blockade of gap junctional communication by heptanol led to a dose-dependent decrease in multinuclearity, suggesting that gap junctional communication precedes cell fusion of human osteoclasts. Indeed, we found a particularly strong expression of Cx43 in the giant osteoclasts of patients with Paget's disease and giant cell tumour of the bone. These results show that gap junctional communication is important for fusion of human mononuclear precursor cells to osteoclasts and that gap junctional Cx43 might play a role in the regulation of size and multinuclearity of human osteoclasts in vivo.