BRAF/MAPK and GSK3 signaling converges to control MITF nuclear export.

The close integration of the MAPK, PI3K, and WNT signaling pathways underpins much of development and is deregulated in cancer. In principle, combinatorial posttranslational modification of key lineage-specific transcription factors would be an effective means to integrate critical signaling events. Understanding how this might be achieved is central to deciphering the impact of microenvironmental cues in development and disease. The microphthalmia-associated transcription factor MITF plays a crucial role in the development of melanocytes, the retinal pigment epithelium, osteoclasts, and mast cells and acts as a lineage survival oncogene in melanoma. MITF coordinates survival, differentiation, cell-cycle progression, cell migration, metabolism, and lysosome biogenesis. However, how the activity of this key transcription factor is controlled remains poorly understood. Here, we show that GSK3, downstream from both the PI3K and Wnt pathways, and BRAF/MAPK signaling converges to control MITF nuclear export. Phosphorylation of the melanocyte MITF-M isoform in response to BRAF/MAPK signaling primes for phosphorylation by GSK3, a kinase inhibited by both PI3K and Wnt signaling. Dual phosphorylation, but not monophosphorylation, then promotes MITF nuclear export by activating a previously unrecognized hydrophobic export signal. Nonmelanocyte MITF isoforms exhibit poor regulation by MAPK signaling, but instead their export is controlled by mTOR. We uncover here an unanticipated mode of MITF regulation that integrates the output of key developmental and cancer-associated signaling pathways to gate MITF flux through the import-export cycle. The results have significant implications for our understanding of melanoma progression and stem cell renewal.

Hypoxia-inducible factor 1-alpha does not regulate osteoclastogenesis but enhances bone resorption activity via prolyl-4-hydroxylase 2.

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 osteoclasts. Phd2+/- murine osteoclasts also exhibited enhanced bone resorption, associated with increased expression of resorption-associated Acp5, in comparison with wild-type cells from littermate controls. Phd3-/- bone marrow precursors displayed accelerated early fusion, mirroring results with HIF-1α siRNA. In vivo, Phd2+/- and Phd3-/- mice exhibited reduced trabecular bone mass, associated with reduced mineralization by Phd2+/- osteoblasts. These data indicate that HIF predominantly functions as a regulator of osteoclast-mediated bone resorption, with little effect on osteoclast differentiation. Inhibition of HIF might therefore represent an alternative strategy to treat diseases characterized by pathological levels of osteolysis. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Hypoxia-Induced Fibroblast Growth Factor 11 Stimulates Osteoclast-Mediated Resorption of Bone.

Over-activation of osteoclasts is directly responsible for pathological bone loss in conditions such as rheumatoid arthritis and cancer metastasis to bone. Hypoxia is a common feature of these conditions, associated with poor prognosis, which also stimulates osteoclast-mediated bone resorption via induction of the hypoxia-inducible transcription factor HIF-1α. Here, we investigate the effects of fibroblast growth factor 11 (FGF11) on osteoclast function. FGF11 is an intracellular FGF that was induced both by hypoxia (2% O2, p < 0.01) and by inhibition of the HIF-regulating prolyl hydroxylase enzymes (CoCl2, p < 0.001) in osteoclasts. Isoform-specific siRNA demonstrated that the induction of Fgf11 mRNA expression by hypoxia is HIF-1α-dependent (p < 0.01). Hypoxic stimulation of bone resorption was inhibited in osteoclasts treated with siRNA targeting FGF11 (p < 0.05). This was at least partially due to reduced secretion of an unidentified pro-resorptive factor downstream of FGF11. FGF11 expression within hypoxic, resorbing osteoclasts co-localised with microtubule-associated alpha-tubulin. FGF11 was also abundantly expressed in osteoclasts within the rheumatoid synovium and in giant cell tumour of bone. This study suggests FGF11 as a novel factor driving pathological bone resorption in osteolytic disease and as a potential target for the development of new anti-resorptive therapeutic agents.

Differential regulation of HIF-mediated pathways increases mitochondrial metabolism and ATP production in hypoxic osteoclasts.

Inappropriate osteoclast activity instigates pathological bone loss in rheumatoid arthritis. We have investigated how osteoclasts generate sufficient ATP for the energy-intensive process of bone resorption in the hypoxic microenvironment associated with this rheumatic condition. We show that in human osteoclasts differentiated from CD14(+) monocytes, hypoxia (24 h, 2% O2 ): (a) increases ATP production and mitochondrial electron transport chain activity (Alamar blue, O2 consumption); (b) increases glycolytic flux (glucose consumption, lactate production); and (c) increases glutamine consumption. We demonstrate that glucose, rather than glutamine, is necessary for the hypoxic increase in ATP production and also for cell survival in hypoxia. Using siRNA targeting specific isoforms of the hypoxia-inducible transcription factor HIF (HIF-1α, HIF-2α), we show that employment of selected components of the HIF-1α-mediated metabolic switch to anaerobic respiration enables osteoclasts to rapidly increase ATP production in hypoxia, while at the same time compromising long-term survival. We propose this atypical HIF-driven metabolic pathway to be an adaptive mechanism to permit rapid bone resorption in the short term while ensuring curtailment of the process in the absence of re-oxygenation.

Use of oxygen-loaded nanobubbles to improve tissue oxygenation: Bone-relevant mechanisms of action and effects on osteoclast differentiation.

Gas-loaded nanobubbles have potential as a method of oxygen delivery to increase tumour oxygenation and therapeutically alleviate tumour hypoxia. However, the mechanism(s) whereby oxygen-loaded nanobubbles increase tumour oxygenation are unknown; with their calculated oxygen-carrying capacity being insufficient to explain this effect. Intra-tumoural hypoxia is a prime therapeutic target, at least partly due to hypoxia-dependent stimulation of the formation and function of bone-resorbing osteoclasts which establish metastatic cells in bone. This study aims to investigate potential mechanism(s) of oxygen delivery and in particular the possible use of oxygen-loaded nanobubbles in preventing bone metastasis via effects on osteoclasts. Lecithin-based nanobubbles preferentially interacted with phagocytic cells (monocytes, osteoclasts) via a combination of lipid transfer, clathrin-dependent endocytosis and phagocytosis. This interaction caused general suppression of osteoclast differentiation via inhibition of cell fusion. Additionally, repeat exposure to oxygen-loaded nanobubbles inhibited osteoclast formation to a greater extent than nitrogen-loaded nanobubbles. This gas-dependent effect was driven by differential effects on the fusion of mononuclear precursor cells to form pre-osteoclasts, partly due to elevated potentiation of RANKL-induced ROS by nitrogen-loaded nanobubbles. Our findings suggest that oxygen-loaded nanobubbles could represent a promising therapeutic strategy for cancer therapy; reducing osteoclast formation and therefore bone metastasis via preferential interaction with monocytes/macrophages within the tumour and bone microenvironment, in addition to known effects of directly improving tumour oxygenation.

Mature primary human osteocytes in mini organotypic cultures secrete FGF23 and PTH1-34-regulated sclerostin.

Introduction: For decades, functional primary human osteocyte cultures have been crucially needed for understanding their role in bone anabolic processes and in endocrine phosphate regulation via the bone-kidney axis. Mature osteocyte proteins (sclerostin, DMP1, Phex and FGF23) play a key role in various systemic diseases and are targeted by successful bone anabolic drugs (anti-sclerostin antibody and teriparatide (PTH1-34)). However, cell lines available to study osteocytes produce very little sclerostin and low levels of mature osteocyte markers. We have developed a primary human 3D organotypic culture system that replicates the formation of mature osteocytes in bone. Methods: Primary human osteoblasts were seeded in a fibrinogen / thrombin gel around 3D-printed hanging posts. Following contraction of the gel around the posts, cells were cultured in osteogenic media and conditioned media was collected for analysis of secreted markers of osteocyte formation. Results: The organoids were viable for at least 6 months, allowing co-culture with different cell types and testing of bone anabolic drugs. Bulk RNAseq data displayed the developing marker trajectory of ossification and human primary osteocyte formation in vitro over an initial 8- week period. Vitamin D3 supplementation increased mineralization and sclerostin secretion, while hypoxia and PTH1-34 modulated sclerostin. Our culture system also secreted FGF23, enabling the future development of a bone-kidney-parathyroid-vascular multi-organoid or organ-on-a-chip system to study disease processes and drug effects using purely human cells. Discussion: This 3D organotypic culture system provides a stable, long-lived, and regulated population of mature human primary osteocytes for a variety of research applications.

VEGF, FLT3 ligand, PlGF and HGF can substitute for M-CSF to induce human osteoclast formation: implications for giant cell tumour pathobiology.

Giant cell tumour of bone (GCTB) is a primary bone tumour that contains numerous very large, hyper-nucleated osteoclastic giant cells. Osteoclasts form from CD14+ monocytes and macrophages in the presence of receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage-colony stimulating factor (M-CSF). GCTB contains numerous growth factors, some of which have been reported to influence osteoclastogenesis and resorption. We investigated whether these growth factors are capable of substituting for M-CSF to support osteoclast formation from cultured human monocytes and whether they influence osteoclast cytomorphology and resorption. Vascular endothelial growth factor-A (VEGF-A), VEGF-D, FLT3 ligand (FL), placental growth factor (PlGF) and hepatocyte growth factor (HGF) supported RANKL-induced osteoclastogenesis in the absence of M-CSF, resulting in the formation of numerous TRAP+ multinucleated cells capable of lacunar resorption. Monocytes cultured in the presence of M-CSF, HGF, VEGF-A and RANKL together resulted in the formation of very large, hyper-nucleated (GCTB-like) osteoclasts that were hyper-resorptive. M-CSF and M-CSF substitute growth factors were identified immunohistochemically in GCTB tissue sections and these factors stimulated the resorption of osteoclasts derived from a subset of GCTBs. Our findings indicate that there are growth factors that are capable of substituting for M-CSF to induce human osteoclast formation and that these factors are present in GCTB where they influence osteoclast cytomorphology and have a role in osteoclast formation and resorption activity.

CD14- mononuclear stromal cells support (CD14+) monocyte-osteoclast differentiation in aneurysmal bone cyst.

Aneurysmal bone cyst (ABC) is a benign osteolytic bone lesion in which there are blood-filled spaces separated by fibrous septa containing giant cells. The nature of the giant cells in this lesion and the mechanism of bone destruction in ABC is not certain. In this study, we have analysed several characteristics of mononuclear and multinucleated cells in the ABC and examined the cellular and molecular mechanisms of ABC osteolysis. The antigenic and functional phenotype of giant cells in ABC was determined by histochemistry/immunohistochemistry using antibodies to macrophage and osteoclast markers. Giant cells and CD14+ and CD14- mononuclear cells were isolated from ABC specimens and cultured on dentine slices and coverslips with receptor activator of nuclear factor κB ligand (RANKL)+/- macrophage-colony stimulating factor (M-CSF) and functional and cytochemical evidence of osteoclast differentiation sought. Giant cells in ABC expressed an osteoclast-like phenotype (CD51+, CD14-, cathepsin K+, TRAP+) and were capable of lacunar resorption, which was inhibited by zoledronate, calcitonin and osteoprotegerin (OPG). When cultured with RANKL±M-CSF, CD14+, but not CD14-, mononuclear cells differentiated into TRAP+ multinucleated cells that were capable of lacunar resorption. M-CSF was not necessary for osteoclast formation from CD14+ cell cultures. CD14- cells variably expressed RANKL, OPG and M-CSF but supported osteoclast differentiation. Our findings show that the giant cells in ABC express an osteoclast-like phenotype and are formed from CD14+ macrophage precursors. CD14- mononuclear stromal cells express osteoclastogenic factors and most likely interact with CD14+ cells to form osteoclast-like giant cells by a RANKL-dependent mechanism.

Ewing sarcoma cells express RANKL and support osteoclastogenesis.

Ewing sarcoma (ES) is a primary malignant round cell tumour of bone characterized by rapid and extensive osteolysis. Cellular mechanisms underlying the rapid bone resorption in ES have not been characterized. Osteoclasts are marrow-derived multinucleated cells that effect tumour osteolysis. The role of ES tumour cells in influencing osteoclast formation and/or directly contributing to the osteolysis in ES has not been determined. Using a tissue culture bioassay, we found that lacunar resorption is not carried out by (CD99(+) ) ES tumour cells, but by (CD68(+) ) macrophage/osteoclast-like cells; this resorption occurred in the absence of the osteoclastogenic factor, receptor activator of nuclear factor κB ligand (RANKL). ES cell lines cultured directly on dentine slices did not resorb the mineral or organic components of the bone matrix. Immunohistochemistry of ES tissue microarrays, western blotting, and RT-PCR studies showed that ES cells strongly expressed both RANKL and macrophage-colony stimulating factor (M-CSF), two major osteoclastogenic factors. When co-cultured with human monocytes, ES cells induced the formation of TRAP(+) osteoclastic cells. Conditioned medium from cultured ES cells did not result in osteoclast formation, indicating that cell-cell contact is required for ES-induced osteoclastogenesis. Our findings indicate that ES cells do not resorb bone directly but that they may support osteoclast formation by a RANKL-dependent mechanism.

Osteoclast formation and function in pigmented villonodular synovitis.

Pigmented villonodular synovitis (PVNS) is a synovial tumour-like lesion that frequently causes osteolysis. PVNS contains numerous macrophages and osteoclast-like giant cells. In this study, we have analysed the cytochemical and functional characteristics of mononuclear and multinucleated cells in PVNS and determined the cellular and humoral mechanisms underlying giant cell formation and resorption in PVNS. Giant cells and CD14(+) and CD14(-) mononuclear cell populations were isolated from PVNS synovial tissue and cultured alone or in the presence and absence of the osteoclastogenic factors, RANKL and M-CSF. Osteoclast formation and activity was assessed by expression of TRAP and evidence of lacunar resorption. Giant cells in PVNS expressed an osteoclast-phenotype (CD51(+) , TRAP(+) , CD14(-) , HLA-DR(-) ) and were formed only in cultures of mononuclear cells that expressed the macrophage marker CD14. Osteoclast formation required RANKL and occurred in both the presence and absence of exogenous M-CSF. CD14(-) cells in PVNS expressed RANKL. Lacunar resorption by PVNS-derived giant cells was abolished by the addition of the bisphosphonate, zoledronate. Our findings indicate that osteoclasts form by a RANKL-dependent mechanism from CD14(+) mononuclear phagocytes in PVNS. Osteoclast formation occurred even in the absence of exogenous M-CSF, a finding which is in keeping with over-expression of M-CSF playing a pathogenic role in this condition. Anti-osteoclast resorptive treatment may be useful to control osteolysis in PVNS.

A New Method to Sort Differentiating Osteoclasts into Defined Homogeneous Subgroups.

Osteoclasts regulate skeletal development but also drive pathological osteolysis, making them prime therapeutic targets. Osteoclast research is limited by the heterogeneity of osteoclast populations generated in vitro, where the mixture of undifferentiated monocytes, binuclear pre-osteoclasts and multinucleated osteoclasts has by necessity been considered a single osteoclast population. This study describes the differentiation of primary human CD14+ monocyte-derived osteoclasts in 3D collagen gels. These osteoclasts remained small (>95% with ≤5 nuclei) but were viable and active; when released from the gel with collagenase, they fused rapidly when reseeded onto solid substrates and resorbed dentine for 2-3 weeks. 3D-generated osteoclasts expressed cell surface markers of osteoclast differentiation (e.g., CD9, RANK, OSCAR, CD63, CD51/61) which, with their small size, enabled live cell sorting of highly enriched viable subpopulations of human osteoclasts that retained full functional resorption capacity. Low-yield osteoclast preparations were strongly enriched to remove undifferentiated cells (e.g., 13.3% CD51/61+ to 84.2% CD51/61+), and subpopulations of CD9+CD51/61- early osteoclasts and CD9+CD51/61+ mature cells were distinguished. This novel approach allows the study of selected populations of differentiating osteoclasts in vitro and opens the door to in-depth transcriptomic and proteomic analysis of these cells, increasing our ability to study human osteoclast molecular mechanisms relevant to development, aging and disease.

Aberrant paracrine signalling for bone remodelling underlies the mutant histone-driven giant cell tumour of bone.

Oncohistones represent compelling evidence for a causative role of epigenetic perturbations in cancer. Giant cell tumours of bone (GCTs) are characterised by a mutated histone H3.3 as the sole genetic driver present in bone-forming osteoprogenitor cells but absent from abnormally large bone-resorbing osteoclasts which represent the hallmark of these neoplasms. While these striking features imply a pathogenic interaction between mesenchymal and myelomonocytic lineages during GCT development, the underlying mechanisms remain unknown. We show that the changes in the transcriptome and epigenome in the mesenchymal cells caused by the H3.3-G34W mutation contribute to increase osteoclast recruitment in part via reduced expression of the TGFß-like soluble factor, SCUBE3. Transcriptional changes in SCUBE3 are associated with altered histone marks and H3.3G34W enrichment at its enhancer regions. In turn, osteoclasts secrete unregulated amounts of SEMA4D which enhances proliferation of mutated osteoprogenitors arresting their maturation. These findings provide a mechanism by which GCTs undergo differentiation in response to denosumab, a drug that depletes the tumour of osteoclasts. In contrast, hTERT alterations, commonly found in malignant GCT, result in the histone-mutated neoplastic cells being independent of osteoclasts for their proliferation, predicting unresponsiveness to denosumab. We provide a mechanism for the initiation of GCT, the basis of which is dysfunctional cross-talk between bone-forming and bone-resorbing cells. The findings highlight the role of tumour/microenvironment bidirectional interactions in tumorigenesis and how this is exploited in the treatment of GCT.

Epidermal growth factor receptor signalling contributes to osteoblastic stromal cell proliferation, osteoclastogenesis and disease progression in giant cell tumour of bone.

AIMS: Epidermal growth factor receptor (EGFR) is implicated in bone remodelling. The aim was to determine whether EGFR protein expression contributes to the aggressiveness and recurrence potential of giant cell tumour of bone (GCTB), an osteolytic primary bone tumour that can exhibit markedly variable clinical behaviour. METHODS AND RESULTS: Immunohistochemical analysis on tissue microarrays (TMA) of 231 primary, 97 recurrent, 17 metastatic and 26 malignant GCTBs was performed using TMA analysis software and whole digital slides allowing validated scoring. EGFR expression was restricted to neoplastic stromal cells and was significantly more frequent in recurrent (71 of 92; 77%) than in non-recurrent GCTBs (86 of 162; 53%) (P = 0.002); and in clinicoradiologically aggressive (31 of 43; 72%) than latent (27 of 54; 50%) cases (P = 0.034). Detecting phosphotyrosine epitopes pY1068 and -pY1173 indicated active EGFR signalling, and finding EGFR ligands EGF and transforming growth factor-α restricted to cells of the monocytic lineage suggested paracrine EGFR activation in stromal cells. In functional studies EGF supported proliferation of GCTB stromal cells, and the addition of EGF and macrophage-colony stimulating factor promoted osteoclastogenesis. CONCLUSION: In GCTB, EGFR signalling in neoplastic stromal cells may contribute to disease progression through promoting stromal cell proliferation and osteoclastogenesis.

Hypoxia-inducible factor regulates osteoclast-mediated bone resorption: role of angiopoietin-like 4.

Hypoxia and the hypoxia-inducible factor (HIF) transcription factor regulate angiogenic-osteogenic coupling and osteoclast-mediated bone resorption. To determine how HIF might coordinate osteoclast and osteoblast function, we studied angiopoietin-like 4 (ANGPTL4), the top HIF target gene in an Illumina HumanWG-6 v3.0 48k array of normoxic vs. hypoxic osteoclasts differentiated from human CD14(+) monocytes (14.3-fold induction, P<0.0004). ANGPTL4 mRNA and protein were induced by 24 h at 2% O(2) in human primary osteoclasts, monocytes, and osteoblasts. ANGPTL4 protein was observed by immunofluorescence in osteoclasts and osteoblasts in vivo. Normoxic inducers of HIF (CoCl(2), desferrioxamine, and l-mimosine) and 100 ng/ml ANGPTL4 stimulated osteoclastic resorption 2- to 3-fold in assays of lacunar dentine resorption, without affecting osteoclast viability. Isoform-specific HIF-1α small interfering RNA ablated hypoxic induction of ANGPTL4 and of resorption, which was rescued by addition of exogenous ANGPTL4 (P<0.001). In the osteoblastic Saos2 cell line, ANGPTL4 caused a dose-dependent increase in proliferation (P<0.01, 100 ng/ml) and, at lower doses (1-25 ng/ml), mineralization. These results demonstrate that HIF is sufficient to enhance osteoclast-mediated bone resorption and that ANGPTL4 can compensate for HIF-1α deficiency with respect to stimulation of osteoclast activity and also augments osteoblast proliferation and differentiation.

Loss of mutual protection between human osteoclasts and chondrocytes in damaged joints initiates osteoclast-mediated cartilage degradation by MMPs.

Osteoclasts are multinucleated, bone-resorbing cells. However, they also digest cartilage during skeletal maintenance, development and in degradative conditions including osteoarthritis, rheumatoid arthritis and primary bone sarcoma. This study explores the mechanisms behind the osteoclast-cartilage interaction. Human osteoclasts differentiated on acellular human cartilage expressed osteoclast marker genes (e.g. CTSK, MMP9) and proteins (TRAP, VNR), visibly damaged the cartilage surface and released glycosaminoglycan in a contact-dependent manner. Direct co-culture with chondrocytes during differentiation increased large osteoclast formation (p < 0.0001) except when co-cultured on dentine, when osteoclast formation was inhibited (p = 0.0002). Osteoclasts cultured on dentine inhibited basal cartilage degradation (p = 0.012). RNA-seq identified MMP8 overexpression in osteoclasts differentiated on cartilage versus dentine (8.89-fold, p = 0.0133), while MMP9 was the most highly expressed MMP. Both MMP8 and MMP9 were produced by osteoclasts in osteosarcoma tissue. This study suggests that bone-resident osteoclasts and chondrocytes exert mutually protective effects on their 'native' tissue. However, when osteoclasts contact non-native cartilage they cause degradation via MMPs. Understanding the role of osteoclasts in cartilage maintenance and degradation might identify new therapeutic approaches for pathologies characterized by cartilage degeneration.

Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor.

BACKGROUND: Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma. METHODS: HIF-1alpha and HIF-2alpha immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration. RESULTS: 17/56 Ewing's tumours were HIF-1alpha-positive, 15 HIF-2alpha-positive and 10 positive for HIF-1alpha and HIF-2alpha. Expression of HIF-1alpha and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1alpha and HIF-2alpha in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2alpha in Ewing's. Downstream transcription was HIF-1alpha-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by >or= 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration. CONCLUSIONS: Co-localisation of HIF-1alpha and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in in vivo induction of HIF. In vitro data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.

Acute hypoxia and osteoclast activity: a balance between enhanced resorption and increased apoptosis.

Osteoclasts are the primary mediators of pathological bone resorption in many conditions in which micro-environmental hypoxia is associated with disease progression. However, effects of hypoxia on human osteoclast activity have not been reported. Mature human osteoclasts were differentiated from peripheral blood or obtained from giant cell tumour of bone. Osteoclasts were exposed to a constant hypoxic environment and then assessed for parameters including resorption (toluidine blue staining of dentine slices), membrane integrity (trypan blue exclusion), apoptosis (TUNEL, DAPI), and osteolysis-associated enzyme activity (TRAP, cathepsin K). 24 h exposure to 2% O(2) produced a 2.5-fold increase in resorption associated with increased TRAP and cathepsin K enzyme activity. Hypoxia-Inducible Factor-1alpha (HIF-1alpha) siRNA completely ablated the hypoxic increase in osteoclast resorption. 24 h at 2% O(2) also increased the number of osteoclasts with compromised membrane integrity from 6% to 21%, with no change in the total osteoclast number or the proportion of late-stage apoptotic cells. Transient reoxygenation returned the percentage of trypan blue-positive cells to normoxic levels, suggesting that osteoclasts can recover from the early stages of cell death. Repeated over an extended period, hypoxia/reoxygenation enhanced osteoclast differentiation at this pO(2). These data suggest that in diseased bone, where the pO(2) may fall to

Distinct roles for the hypoxia-inducible transcription factors HIF-1α and HIF-2α in human osteoclast formation and function.

Bone homeostasis is maintained by a balance between osteoblast-mediated bone formation and osteoclast-driven bone resorption. Hypoxia modulates this relationship partially via direct and indirect effects of the hypoxia-inducible factor-1 alpha (HIF-1α) transcription factor on osteoclast formation and bone resorption. Little data is available on the role(s) of the HIF-2α isoform of HIF in osteoclast biology. Here we describe induction of HIF-1α and HIF-2α during the differentiation of human CD14+ monocytes into osteoclasts. Knockdown of HIF-1α did not affect osteoclast differentiation but prevented the increase in bone resorption that occurs under hypoxic conditions. HIF-2α knockdown did not affect bone resorption but moderately inhibited osteoclast formation. Growth of osteoclasts in 3D gels reversed the effect of HIF-2α knockdown; HIF-2α siRNA increasing osteoclast formation in 3D. Glycolysis is the main HIF-regulated pathway that drives bone resorption. HIF knockdown only affected glucose uptake and bone resorption in hypoxic conditions. Inhibition of glycolysis with 2-deoxy-D-glucose (2-DG) reduced osteoclast formation and activity under both basal and hypoxic conditions, emphasising the importance of glycolytic metabolism in osteoclast biology. In summary, HIF-1α and HIF-2α play different but overlapping roles in osteoclast biology, highlighting the importance of the HIF pathway as a potential therapeutic target in osteolytic disease.

Osteoblast-Osteoclast Coculture Amplifies Inhibitory Effects of FG-4592 on Human Osteoclastogenesis and Reduces Bone Resorption.

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 stabilize HIF protein, introduces the potential for a new clinical strategy to treat osteolytic conditions such as osteoporosis, osteonecrosis, and skeletal fracture and nonunion. However, bone-resorbing osteoclasts also play a central role in bone remodeling and pathological osteolysis, and HIF promotes osteoclast activation and bone loss in vitro. It is therefore likely that the result 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 stabilized HIF protein and stimulated osteoclast-mediated bone resorption, but inhibited differentiation of human CD14+ monocytes into osteoclasts. Formation of osteoclasts in a more physiologically relevant 3D collagen gel did not affect the sensitivity of osteoclastogenesis to FG-4592, but increased sensitivity to reduced concentrations of RANKL. Coculture with osteoblasts amplified inhibition of osteoclastogenesis by FG-4592, whether the osteoblasts were proliferating, differentiating, or in the presence of exogenous M-CSF and RANKL. Osteoblast coculture dampened the ability of high concentrations of FG-4592 to increase bone resorption. These data provide support for the therapeutic use of PHD enzyme inhibitors to improve bone formation and/or reduce bone loss for the treatment of osteolytic pathologies and indicate that FG-4592 might act in vivo to inhibit the formation and activity of the osteoclasts that drive osteolysis. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

The Adenosine A2B Receptor Drives Osteoclast-Mediated Bone Resorption in Hypoxic Microenvironments.

Osteoclast-mediated bone destruction is amplified in the hypoxic synovial microenvironment of rheumatoid arthritis (RA). This increased bone resorption is driven by the hypoxia-inducible transcription factor HIF. We identified hypoxic induction of the HIF-regulated adenosine A2B receptor in primary human osteoclasts (mRNA, 3.8-fold increase, p < 0.01) and sought to identify the role(s) of purinergic signaling via this receptor in the bone resorption process. Primary human osteoclasts were differentiated from CD14+ monocytes and exposed to hypoxia (2% O2) and A2B receptor inhibitors (MRS1754, PSB603). The hypoxic increase in bone resorption was prevented by the inhibition of the A2B receptor, at least partly by the attenuation of glycolytic and mitochondrial metabolism via inhibition of HIF. A2B receptor inhibition also reduced osteoclastogenesis in hypoxia by inhibiting early cell fusion (day 3-4, p < 0.05). The A2B receptor is only functional in hypoxic or inflammatory environments when the extracellular concentrations of adenosine (1.6-fold increase, p < 0.05) are sufficient to activate the receptor. Inhibition of the A2B receptor under normoxic conditions therefore did not affect any parameter tested. Reciprocal positive regulation of HIF and the A2B receptor in a hypoxic microenvironment thus enhances glycolytic and mitochondrial metabolism in osteoclasts to drive increased bone resorption. A2B receptor inhibition could potentially prevent the pathological osteolysis associated with hypoxic diseases such as rheumatoid arthritis.