Osteoblast–Osteoclast Coculture Amplifies Inhibitory Effects of FG ‐4592 on Human Osteoclastogenesis and Reduces Bone Resorption (original) (raw)
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2019
The link between bone and blood vessels is regulated by hypoxia and the hypoxia-inducible transcription factor, HIF, which drives both osteogenesis and angiogenesis. The recent clinical approval of PHD enzyme inhibitors, which stabilise HIF protein, introduces the potential for a new clinical strategy to treat osteolytic conditions such as osteoporosis, osteonecrosis and skeletal fracture and non-union. However, bone-resorbing osteoclasts also play a central role in bone remodelling and pathological osteolysis and HIF promotes osteoclast activation and bone loss in vitro. It is therefore likely that the final outcome of PHD enzyme inhibition in vivo would be mediated by a balance between increased bone formation and increased bone resorption. It is essential that we improve our understanding of the effects of HIF on osteoclast formation and function, and consider the potential contribution of inhibitory interactions with other musculoskeletal cells.The PHD enzyme inhibitor FG-4592 s...
Regulation of Osteoclast Formation and Function
Reviews in Endocrine & Metabolic Disorders, 2001
The human skeleton is continuously remodeling during growth and in response to mechanical stress and hormonal regulation. The maintenance of normal bone mass during adult life depends on a precise balance between osteoblastic bone formation and osteoclastic bone destruction, implicating tight coupling between these two cellular activities. In various skeletal diseases associated with bone loss, including osteoporosis, hypercalcemia of malignancy,
A novel osteoblast-derived molecule that inhibits the early phase of osteoclast formation
Bone, 2000
We have cloned and expressed murine osteoclast inhibitory lectin (mOCIL), a 207-amino acid type II transmembrane C-type lectin. In osteoclast formation assays of primary murine calvarial osteoblasts with bone marrow cells, antisense oligonucleotides for mOCIL increased tartrate-resistant acid phosphatase-positive mononucleate cell formation by 3-5-fold, whereas control oligonucleotides had no effect. The extracellular domain of mOCIL, expressed as a recombinant protein in Escherichia coli, dose-dependently inhibited multinucleate osteoclast formation in murine osteoblast and spleen cell co-cultures as well as in spleen cell cultures treated with RANKL and macrophage colony-stimulating factor. Furthermore, mOCIL acted directly on macrophage/monocyte cells as evidenced by its inhibitory action on adherent spleen cell cultures, which were depleted of stromal and lymphocytic cells. mOCIL completely inhibited osteoclast formation during the proliferative phase of osteoclast formation and resulted in 70% inhibition during the differentiation phase. Osteoblast OCIL mRNA expression was enhanced by parathyroid hormone, calcitriol, interleukin-1␣ and -11, and retinoic acid. In rodent tissues, Northern blotting, in situ hybridization, and immunohistochemistry demonstrated OCIL expression in osteoblasts and chondrocytes as well as in a variety of extraskeletal tissues. The overlapping tissue distribution of OCIL mRNA and protein with that of RANKL strongly suggests an interaction between these molecules in the skeleton and in extraskeletal tissues.
Journal of translational medicine, 2004
Osteoclasts are cells of hematopoietic origin with a unique property of dissolving bone; their inhibition is a principle for treatment of diseases of bone loss. Protocols for generation of human osteoclasts in vitro have been described, but they often result in cells of low activity, raising questions on cell phenotype and suitability of such assays for screening of bone resorption inhibitors. Here we describe an optimized protocol for the production of stable amounts of highly active human osteoclasts. Mononuclear cells were isolated from human peripheral blood by density centrifugation, seeded at 600,000 cells per 96-well and cultured for 17 days in alpha-MEM medium, supplemented with 10% of selected fetal calf serum, 1 microM dexamethasone and a mix of macrophage-colony stimulating factor (M-CSF, 25 ng/ml), receptor activator of NFkappaB ligand (RANKL, 50 ng/ml), and transforming growth factor-beta1 (TGF-beta1, 5 ng/ml). Thus, in addition to widely recognized osteoclast-generatin...
RANKL/OPG; Critical role in bone physiology
Reviews in Endocrine and Metabolic Disorders, 2015
After it was proposed that the osteoblast lineage controlled the formation of osteoclasts, cell culture methods were developed that established this to be the case. Evidence was obtained that cytokines and hormones that promote osteoclast formation act first on osteoblast lineage cells to promote the production of a membrane-bound regulator of osteoclastogenesis. This proved to be receptor activator of NF-kB ligand (RANKL) a member of the tumor necrosis factor ligand family that acts upon its receptor RANK in the hematopoietic lineage, with interaction restricted by a decoy soluble receptor osteoprotegerin (OPG), also a product of the osteoblast lineage. The physiological roles of these factors were established through genetic and pharmacological studies, have led to a new physiology of bone, with complete revision of older ideas over the last 15 years, ultimately leading to the development of new pharmaceutical agents for bone disease.
Advances in osteoclast biology reveal potential new drug targets and new roles for osteoclasts
Journal of Bone and Mineral Research, 2013
Osteoclasts are multinucleated myeloid lineage cells formed in response to M-CSF and RANKL by fusion of bone marrow-derived precursors that circulate in the blood and are attracted to sites of bone resorption in response to factors, such as sphingosine-1 phosphate signaling. Major advances in understanding of the molecular mechanisms regulating osteoclast functions have been made in the past 20 years mainly from mouse and human genetic studies. These have revealed that osteoclasts express and respond to pro-and anti-inflammatory cytokines. Some of these cytokine activate NF-κB and NFATc1 signaling to induce osteoclast formation and activity and also regulate communication with neighboring cells through signaling proteins, including ephrins and semaphorins. Osteoclasts also positively and negatively regulate immune responses and osteoblastic bone formation. These advances have led to development of new inhibitors of bone resorption that are in clinical use or in clinical trials; and more should follow, based on these advances. This paper reviews current understanding of how bone resorption is regulated both positively and negatively in normal and pathologic states.
2015
The growth, maintenance and repair of bone are regulated by homeostatic interactions between osteoclasts, which resorb bone, and osteoblasts, which produce bone. Disruption of this balance in favor of osteoclast over-activation, in the absence of a balancing amount of bone formation, results in pathological bone loss such as that which occurs in osteoporosis, primary bone cancer, cancer metastasis to bone and rheumatoid arthritis. Hypoxia is a major micro-environmental feature of these conditions which is predictive of disease progression and poor prognosis. There is currently considerable interest in the mechanisms whereby hypoxia, the hypoxia-inducible transcription factors HIF-1α and HIF-2α, and the HIF-regulating prolyl hydroxylase (PHD) enzymes affect bone re-modelling and bone pathologies. This review summarises the evidence for HIF-mediated stimulation of osteogenicangiogenic coupling and the use of PHD inhibitors to stimulate new bone formation and prevent osteolytic disease...
The Journal of pathology, 2017
Osteogenic-angiogenic coupling is promoted by the hypoxia-inducible factor 1-alpha (HIF-1α) transcription factor, provoking interest in HIF activation as a therapeutic strategy to improve osteoblast mineralization and treat pathological osteolysis. However, HIF also enhances the bone-resorbing activity of mature osteoclasts. It is therefore essential to determine the full effect(s) of HIF on both the formation and the bone-resorbing function of osteoclasts in order to understand how they might respond to such a strategy. Expression of HIF-1α mRNA and protein increased during osteoclast differentiation from CD14+ monocytic precursors, additionally inducing expression of the HIF-regulated glycolytic enzymes. However, HIF-1α siRNA only moderately affected osteoclast differentiation, accelerating fusion of precursor cells. HIF induction by inhibition of the regulatory prolyl-4-hydroxylase (PHD) enzymes reduced osteoclastogenesis, but was confirmed to enhance bone resorption by mature os...
Molecular Medicine Reports, 2012
The aim of the present study was to compare the osteoclast-inhibiting ability of recombinant osteoprotegerin (OPG) protein (rhOPG-Fc) and recombinant receptor activator of nuclear factor κB (rhRANK) in vitro and in vivo. Osteoclasts were cultured with either rhOPG-Fc or rhRANK for 9 days. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and resorption pits in bone slices were then counted. In the in vivo investigation, female mice were bilaterally ovariectomized (OVX) and intraperitoneally injected with 3 mg/kg rhOPG-Fc or rhRANK for 12 weeks, respectively. Bone metabolism, bone mineral density and microstructure changes were then evaluated. The number of TRAP-positive cells and bone resorption pits decreased significantly following culture with either rhOPG-Fc or rhRANK, and this was more marked following culture with rhRANK compared with rhOPG-Fc. The levels of calcium and alkaline phosphatase in the serum were similar pre-OVX and after 12 weeks of treatment, while the levels of phosphorus in the serum were higher following treatment with rhRANK compared with rhOPG. The bone mineral density (BMD) of the whole body, femoral neck and L4 lumbar vertebral body in the mice treated with either rhOPG-Fc or rhRANK increased markedly. In addition, the mice treated with rhRANK exhibited significantly higher BMD in the femoral neck and lumbar vertebral body compared with those treated with rhOPG-Fc. Microcomputed tomography analysis demonstrated that the mice treated with rhRANK exhibited an increased bone volume and structure model index, and decreased trabecular spacing compared with those treated with rhOPG-Fc. rhRANK increased the inhibition of osteoclast differentiation and bone resorption, and rescued OVX-induced osteoporosis more effectively compared with rhOPG-Fc.
Inhibitors of histone deacetylases in class I and class II suppress human osteoclasts in vitro
Journal of Cellular Physiology, 2011
Histone deacetylase inhibitors (HDACi) suppress cancer cell growth, inflammation, and bone resorption. The aim of this study was to determine the effect of inhibitors of different HDAC classes on human osteoclast activity in vitro. Human osteoclasts generated from blood mononuclear cells stimulated with receptor activator of nuclear factor kappa B (RANK) ligand were treated with a novel compound targeting classes I and II HDACs (1179.4b), MS-275 (targets class I HDACs), 2664.12 (targets class II HDACs), or suberoylanilide hydroxamic acid (SAHA; targets classes I and II HDACs). Osteoclast differentiation was assessed by expression of tartrate resistant acid phosphatase and resorption of dentine. Expression of mRNA encoding for osteoclast genes including RANK, calcitonin receptor (CTR), c-Fos, tumur necrosis factor (TNF) receptor associated factor (TRAF)6, nuclear factor of activated T cells (NFATc1), interferon-b, TNF-like weak inducer of apoptosis (TWEAK), and osteoclast-associated receptor (OSCAR) were assessed. Expression of HDACs 1-10 during osteoclast development was also assessed. 1179.4b significantly reduced osteoclast activity (IC 50 < 0.16 nM). MS-275 (IC 50 54.4 nM) and 2664.12 (IC 50 > 100 nM) were markedly less effective. A combination of MS-275 and 2664.12 inhibited osteoclast activity similar to 1179.4b (IC 50 0.35 nM). SAHA was shown to suppress osteoclast activity (IC 50 12 nM). 1179.4b significantly ( P < 0.05) reduced NFATc1, CTR, and OSCAR expression during the later stages of osteoclast development. Class I HDAC 8 and Class II HDAC5 were both elevated ( P < 0.05) during osteoclast development. Results suggest that inhibition of both classes I and II HDACs may be required to suppress human osteoclastic bone resorption in vitro. ORIGINAL RESEARCH ARTICLE 3233 J o u r n a l o f J o u r n a l o f Cellular Physiology Cellular Physiology ß 2 0 1 1 W I L E Y P E R I O D I C A L S , I N C .