Helen Knowles - Academia.edu (original) (raw)

Papers by Helen Knowles

Research paper thumbnail of Hypoxia and oxidative stress in breast cancer Hypoxia and tumourigenesis

Breast Cancer Research, Jul 17, 2001

Research paper thumbnail of Mature primary human osteocytes in mini organotypic cultures secrete FGF23 and PTH1-34-regulated sclerostin

Frontiers in Endocrinology

IntroductionFor decades, functional primary human osteocyte cultures have been crucially needed f... more IntroductionFor 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.MethodsPrimary 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.ResultsThe organoids were via...

Research paper thumbnail of 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 ca... more 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 TGFβ-like soluble factor, SCUBE3. In turn, osteoclasts secrete unregulated amounts of SEMA4D enhancing proliferation of mutated osteoprogenitors and arresting their maturation. These findings provide a mechanism by which GCTs undergo differentiation upon den...

Research paper thumbnail of The Adenosine A2B Receptor Drives Osteoclast-Mediated Bone Resorption in Hypoxic Microenvironments

Cells, Jun 21, 2019

Osteoclast-mediated bone destruction is amplified in the hypoxic synovial microenvironment of rhe... more 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 A 2B 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% O 2) and A 2B receptor inhibitors (MRS1754, PSB603). The hypoxic increase in bone resorption was prevented by the inhibition of the A 2B receptor, at least partly by the attenuation of glycolytic and mitochondrial metabolism via inhibition of HIF. A 2B receptor inhibition also reduced osteoclastogenesis in hypoxia by inhibiting early cell fusion (day 3-4, p < 0.05). The A 2B 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 A 2B receptor under normoxic conditions therefore did not affect any parameter tested. Reciprocal positive regulation of HIF and the A 2B receptor in a hypoxic microenvironment thus enhances glycolytic and mitochondrial metabolism in osteoclasts to drive increased bone resorption. A 2B receptor inhibition could potentially prevent the pathological osteolysis associated with hypoxic diseases such as rheumatoid arthritis.

Research paper thumbnail of A New Method to Sort Differentiating Osteoclasts into Defined Homogeneous Subgroups

Cells

Osteoclasts regulate skeletal development but also drive pathological osteolysis, making them pri... more 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 function...

Research paper thumbnail of Multiple Roles of Angiopoietin-Like 4 in Osteolytic Disease

Frontiers in Endocrinology, 2017

Hypoxia and the hypoxia-inducible factor (HIF) transcription factor drive pathological bone loss ... more Hypoxia and the hypoxia-inducible factor (HIF) transcription factor drive pathological bone loss in conditions including rheumatoid arthritis (RA), osteoarthritis, osteoporosis, primary bone tumours, and bone metastatic cancer. There is therefore considerable interest in determining the function(s) of HIF-induced genes in these pathologies. Angiopoietin-like 4 (ANGPTL4) is an adipose-derived, HIF-1α-and PPARγ-induced gene that was originally discovered as an endocrine and autocrine/paracrine regulator of lipid metabolism. Given the inverse relationship between bone adiposity and fracture risk, ANGPTL4 might be considered a good candidate for mediating the downstream effects of HIF-1α relevant to osteolytic disease. This review will consider the possible roles of ANGPTL4 in regulation of osteoclast-mediated bone resorption, cartilage degradation, angiogenesis, and inflammation, focusing on results obtained in the study of RA. Possible roles in other musculoskeletal pathologies will also be discussed. This will highlight ANGPTL4 as a regulator of multiple disease processes, which could represent a novel therapeutic target in osteolytic musculoskeletal disease.

Research paper thumbnail of The CXCR4-CXCL12 axis in Ewing sarcoma: promotion of tumor growth rather than metastatic disease

Clinical Sarcoma Research, 2012

Background Chemokine receptor CXCR4, together with its ligand CXCL12, plays critical roles in can... more Background Chemokine receptor CXCR4, together with its ligand CXCL12, plays critical roles in cancer progression, including growth, metastasis and angiogenesis. Ewing sarcoma is a sarcoma with poor prognosis despite current therapies, particularly for patients with advanced-stage disease. Lungs and bone (marrow), organs of predilection for (primary/metastatic) Ewing sarcoma, represent predominant CXCL12 sources. Methods To gain insight into the role of the CXCR4-CXCL12 axis in Ewing sarcoma, CXCR4, CXCL12 and hypoxia-inducible factor-1α protein expression was studied in therapy-naïve and metastatic tumors by immunohistochemistry. CXCR4 function was assessed in vitro, by flow cytometry and proliferation/ cell viability assays, in the presence of recombinant CXCL12 and/or CXCR4-antagonist AMD3100 or under hypoxic conditions. Results Whereas CXCR4 was predominantly expressed by tumor cells, CXCL12 was observed in both tumor and stromal areas. Survival analysis revealed an (expression l...

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

Scientific Reports

Osteoclasts are multinucleated, bone-resorbing cells. However, they also digest cartilage during ... more 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 expre...

Research paper thumbnail of Loss of mutually protective effects between osteoclasts and chondrocytes in damaged joints drives osteoclast-mediated cartilage degradation via matrix metalloproteinases

bioRxiv, 2021

Osteoclasts are large multinucleated cells that resorb bone to regulate bone remodelling during s... more Osteoclasts are large multinucleated cells that resorb bone to regulate bone remodelling during skeletal maintenance and development. It is overlooked that osteoclasts also digest cartilage during this process, as well as in degradative conditions including osteoarthritis, rheumatoid arthritis and primary bone sarcomas such as giant cell tumour of bone. This study explores the poorly understood mechanisms behind the interaction between osteoclasts and cartilage. Morphologically, osteoclasts differentiated on acellular human cartilage formed multinucleated cells expressing characteristic osteoclast marker genes (e.g. CTSK, MMP9) and proteins (TRAP, VNR) that visibly damaged the cartilage surface by SEM, but without the formation of resorption pits. Osteoclasts caused increased glycosaminoglycan (GAG) release from acellular and cellular human cartilage that was dependent on direct contact with the substrate. Direct co-culture with chondrocytes during osteoclast differentiation increas...

Research paper thumbnail of Osteoblast-osteoclast co-culture amplifies inhibitory effects of FG-4592 on osteoclast formation and reduces bone resorption activity

The link between bone and blood vessels is regulated by hypoxia and the hypoxia-inducible transcr... more 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...

Research paper thumbnail of Hypoxic regulation of osteoclast differentiation and bone resorption activity

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

The Journal of pathology, Jul 18, 2017

Osteogenic-angiogenic coupling is promoted by the hypoxia-inducible factor 1-alpha (HIF-1α) trans... more 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...

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

The Journal of Pathology, 2013

Research paper thumbnail of Hypoxia-Inducible Factor (HIF) regulates Osteoclast-Mediated Bone Resorption via Angiopoietin-like 4 (ANGPTL4)

Research paper thumbnail of Hypoxia-inducible factor regulates osteoclast-mediated bone resorption: role of angiopoietin-like 4

The FASEB Journal, 2010

Hypoxia and the hypoxia-inducible factor (HIF) transcription factor regulate angiogenic-osteogeni... more 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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.001). In the osteoblastic Saos2 cell line, ANGPTL4 caused a dose-dependent increase in proliferation (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;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.

Research paper thumbnail of Macrophage infiltration and angiogenesis in human malignancy

Novartis Foundation symposium, 2004

It is well recognized that human tumours are hypoxic compared to normal adjacent tissues and that... more It is well recognized that human tumours are hypoxic compared to normal adjacent tissues and that hypoxia is related to a poor outcome regardless of modality of treatment, including surgery alone, radiotherapy or chemotherapy. Hypoxia regulates a complex programme of gene transcription via hypoxia-inducible factors 1 and 2 (HIF-1, -2). We have shown that in breast cancer and many other tumour types, tumour-associated macrophages express high levels of HIF-2alpha compared to normal tissue macrophages and compared to the tumour. This high macrophage HIF-2alpha is an independent prognostic factor for poor outcome. The mechanisms up-regulating HLF-2alpha in macrophages may include inflammatory cytokines as well as hypoxia. Differentiation of monocytes into macrophages increases the basal level of HIF-2alpha protein and changes the programme of hypoxia. Many of these inducible genes are involved in inflammation and angiogenesis. Thus, the conversion of a peripheral monocyte into a macrop...

Research paper thumbnail of Hypoxia, hypoxia-inducible factor (HIF) and bone homeostasis: focus on osteoclast-mediated bone resorption

The growth, maintenance and repair of bone are regulated by homeostatic interactions between oste... more 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...

Research paper thumbnail of Distinct roles for the hypoxia-inducible transcription factors HIF-1α and HIF-2α in human osteoclast formation and function

Scientific Reports

Bone homeostasis is maintained by a balance between osteoblast-mediated bone formation and osteoc... more 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...

Research paper thumbnail of Osteoblast–Osteoclast Coculture Amplifies Inhibitory Effects of FG ‐4592 on Human Osteoclastogenesis and Reduces Bone Resorption

JBMR Plus

The link between bone and blood vessels is regulated by hypoxia and the hypoxia-inducible transcr... more 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.

Research paper thumbnail of Transcriptomic profiling of the myeloma bone-lining niche reveals BMP signalling inhibition to improve bone disease

Nature Communications

Multiple myeloma is an incurable, bone marrow-dwelling malignancy that disrupts bone homeostasis ... more Multiple myeloma is an incurable, bone marrow-dwelling malignancy that disrupts bone homeostasis causing skeletal damage and pain. Mechanisms underlying myeloma-induced bone destruction are poorly understood and current therapies do not restore lost bone mass. Using transcriptomic profiling of isolated bone lining cell subtypes from a murine myeloma model, we find that bone morphogenetic protein (BMP) signalling is upregulated in stromal progenitor cells. BMP signalling has not previously been reported to be dysregulated in myeloma bone disease. Inhibition of BMP signalling in vivo using either a small molecule BMP receptor antagonist or a solubilized BMPR1a-FC receptor ligand trap prevents trabecular and cortical bone volume loss caused by myeloma, without increasing tumour burden. BMP inhibition directly reduces osteoclastogenesis, increases osteoblasts and bone formation, and suppresses bone marrow sclerostin levels. In summary we describe a novel role for the BMP pathway in myel...

Research paper thumbnail of Hypoxia and oxidative stress in breast cancer Hypoxia and tumourigenesis

Breast Cancer Research, Jul 17, 2001

Research paper thumbnail of Mature primary human osteocytes in mini organotypic cultures secrete FGF23 and PTH1-34-regulated sclerostin

Frontiers in Endocrinology

IntroductionFor decades, functional primary human osteocyte cultures have been crucially needed f... more IntroductionFor 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.MethodsPrimary 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.ResultsThe organoids were via...

Research paper thumbnail of 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 ca... more 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 TGFβ-like soluble factor, SCUBE3. In turn, osteoclasts secrete unregulated amounts of SEMA4D enhancing proliferation of mutated osteoprogenitors and arresting their maturation. These findings provide a mechanism by which GCTs undergo differentiation upon den...

Research paper thumbnail of The Adenosine A2B Receptor Drives Osteoclast-Mediated Bone Resorption in Hypoxic Microenvironments

Cells, Jun 21, 2019

Osteoclast-mediated bone destruction is amplified in the hypoxic synovial microenvironment of rhe... more 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 A 2B 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% O 2) and A 2B receptor inhibitors (MRS1754, PSB603). The hypoxic increase in bone resorption was prevented by the inhibition of the A 2B receptor, at least partly by the attenuation of glycolytic and mitochondrial metabolism via inhibition of HIF. A 2B receptor inhibition also reduced osteoclastogenesis in hypoxia by inhibiting early cell fusion (day 3-4, p < 0.05). The A 2B 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 A 2B receptor under normoxic conditions therefore did not affect any parameter tested. Reciprocal positive regulation of HIF and the A 2B receptor in a hypoxic microenvironment thus enhances glycolytic and mitochondrial metabolism in osteoclasts to drive increased bone resorption. A 2B receptor inhibition could potentially prevent the pathological osteolysis associated with hypoxic diseases such as rheumatoid arthritis.

Research paper thumbnail of A New Method to Sort Differentiating Osteoclasts into Defined Homogeneous Subgroups

Cells

Osteoclasts regulate skeletal development but also drive pathological osteolysis, making them pri... more 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 function...

Research paper thumbnail of Multiple Roles of Angiopoietin-Like 4 in Osteolytic Disease

Frontiers in Endocrinology, 2017

Hypoxia and the hypoxia-inducible factor (HIF) transcription factor drive pathological bone loss ... more Hypoxia and the hypoxia-inducible factor (HIF) transcription factor drive pathological bone loss in conditions including rheumatoid arthritis (RA), osteoarthritis, osteoporosis, primary bone tumours, and bone metastatic cancer. There is therefore considerable interest in determining the function(s) of HIF-induced genes in these pathologies. Angiopoietin-like 4 (ANGPTL4) is an adipose-derived, HIF-1α-and PPARγ-induced gene that was originally discovered as an endocrine and autocrine/paracrine regulator of lipid metabolism. Given the inverse relationship between bone adiposity and fracture risk, ANGPTL4 might be considered a good candidate for mediating the downstream effects of HIF-1α relevant to osteolytic disease. This review will consider the possible roles of ANGPTL4 in regulation of osteoclast-mediated bone resorption, cartilage degradation, angiogenesis, and inflammation, focusing on results obtained in the study of RA. Possible roles in other musculoskeletal pathologies will also be discussed. This will highlight ANGPTL4 as a regulator of multiple disease processes, which could represent a novel therapeutic target in osteolytic musculoskeletal disease.

Research paper thumbnail of The CXCR4-CXCL12 axis in Ewing sarcoma: promotion of tumor growth rather than metastatic disease

Clinical Sarcoma Research, 2012

Background Chemokine receptor CXCR4, together with its ligand CXCL12, plays critical roles in can... more Background Chemokine receptor CXCR4, together with its ligand CXCL12, plays critical roles in cancer progression, including growth, metastasis and angiogenesis. Ewing sarcoma is a sarcoma with poor prognosis despite current therapies, particularly for patients with advanced-stage disease. Lungs and bone (marrow), organs of predilection for (primary/metastatic) Ewing sarcoma, represent predominant CXCL12 sources. Methods To gain insight into the role of the CXCR4-CXCL12 axis in Ewing sarcoma, CXCR4, CXCL12 and hypoxia-inducible factor-1α protein expression was studied in therapy-naïve and metastatic tumors by immunohistochemistry. CXCR4 function was assessed in vitro, by flow cytometry and proliferation/ cell viability assays, in the presence of recombinant CXCL12 and/or CXCR4-antagonist AMD3100 or under hypoxic conditions. Results Whereas CXCR4 was predominantly expressed by tumor cells, CXCL12 was observed in both tumor and stromal areas. Survival analysis revealed an (expression l...

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

Scientific Reports

Osteoclasts are multinucleated, bone-resorbing cells. However, they also digest cartilage during ... more 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 expre...

Research paper thumbnail of Loss of mutually protective effects between osteoclasts and chondrocytes in damaged joints drives osteoclast-mediated cartilage degradation via matrix metalloproteinases

bioRxiv, 2021

Osteoclasts are large multinucleated cells that resorb bone to regulate bone remodelling during s... more Osteoclasts are large multinucleated cells that resorb bone to regulate bone remodelling during skeletal maintenance and development. It is overlooked that osteoclasts also digest cartilage during this process, as well as in degradative conditions including osteoarthritis, rheumatoid arthritis and primary bone sarcomas such as giant cell tumour of bone. This study explores the poorly understood mechanisms behind the interaction between osteoclasts and cartilage. Morphologically, osteoclasts differentiated on acellular human cartilage formed multinucleated cells expressing characteristic osteoclast marker genes (e.g. CTSK, MMP9) and proteins (TRAP, VNR) that visibly damaged the cartilage surface by SEM, but without the formation of resorption pits. Osteoclasts caused increased glycosaminoglycan (GAG) release from acellular and cellular human cartilage that was dependent on direct contact with the substrate. Direct co-culture with chondrocytes during osteoclast differentiation increas...

Research paper thumbnail of Osteoblast-osteoclast co-culture amplifies inhibitory effects of FG-4592 on osteoclast formation and reduces bone resorption activity

The link between bone and blood vessels is regulated by hypoxia and the hypoxia-inducible transcr... more 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...

Research paper thumbnail of Hypoxic regulation of osteoclast differentiation and bone resorption activity

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

The Journal of pathology, Jul 18, 2017

Osteogenic-angiogenic coupling is promoted by the hypoxia-inducible factor 1-alpha (HIF-1α) trans... more 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...

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

The Journal of Pathology, 2013

Research paper thumbnail of Hypoxia-Inducible Factor (HIF) regulates Osteoclast-Mediated Bone Resorption via Angiopoietin-like 4 (ANGPTL4)

Research paper thumbnail of Hypoxia-inducible factor regulates osteoclast-mediated bone resorption: role of angiopoietin-like 4

The FASEB Journal, 2010

Hypoxia and the hypoxia-inducible factor (HIF) transcription factor regulate angiogenic-osteogeni... more 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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.001). In the osteoblastic Saos2 cell line, ANGPTL4 caused a dose-dependent increase in proliferation (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;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.

Research paper thumbnail of Macrophage infiltration and angiogenesis in human malignancy

Novartis Foundation symposium, 2004

It is well recognized that human tumours are hypoxic compared to normal adjacent tissues and that... more It is well recognized that human tumours are hypoxic compared to normal adjacent tissues and that hypoxia is related to a poor outcome regardless of modality of treatment, including surgery alone, radiotherapy or chemotherapy. Hypoxia regulates a complex programme of gene transcription via hypoxia-inducible factors 1 and 2 (HIF-1, -2). We have shown that in breast cancer and many other tumour types, tumour-associated macrophages express high levels of HIF-2alpha compared to normal tissue macrophages and compared to the tumour. This high macrophage HIF-2alpha is an independent prognostic factor for poor outcome. The mechanisms up-regulating HLF-2alpha in macrophages may include inflammatory cytokines as well as hypoxia. Differentiation of monocytes into macrophages increases the basal level of HIF-2alpha protein and changes the programme of hypoxia. Many of these inducible genes are involved in inflammation and angiogenesis. Thus, the conversion of a peripheral monocyte into a macrop...

Research paper thumbnail of Hypoxia, hypoxia-inducible factor (HIF) and bone homeostasis: focus on osteoclast-mediated bone resorption

The growth, maintenance and repair of bone are regulated by homeostatic interactions between oste... more 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...

Research paper thumbnail of Distinct roles for the hypoxia-inducible transcription factors HIF-1α and HIF-2α in human osteoclast formation and function

Scientific Reports

Bone homeostasis is maintained by a balance between osteoblast-mediated bone formation and osteoc... more 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...

Research paper thumbnail of Osteoblast–Osteoclast Coculture Amplifies Inhibitory Effects of FG ‐4592 on Human Osteoclastogenesis and Reduces Bone Resorption

JBMR Plus

The link between bone and blood vessels is regulated by hypoxia and the hypoxia-inducible transcr... more 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.

Research paper thumbnail of Transcriptomic profiling of the myeloma bone-lining niche reveals BMP signalling inhibition to improve bone disease

Nature Communications

Multiple myeloma is an incurable, bone marrow-dwelling malignancy that disrupts bone homeostasis ... more Multiple myeloma is an incurable, bone marrow-dwelling malignancy that disrupts bone homeostasis causing skeletal damage and pain. Mechanisms underlying myeloma-induced bone destruction are poorly understood and current therapies do not restore lost bone mass. Using transcriptomic profiling of isolated bone lining cell subtypes from a murine myeloma model, we find that bone morphogenetic protein (BMP) signalling is upregulated in stromal progenitor cells. BMP signalling has not previously been reported to be dysregulated in myeloma bone disease. Inhibition of BMP signalling in vivo using either a small molecule BMP receptor antagonist or a solubilized BMPR1a-FC receptor ligand trap prevents trabecular and cortical bone volume loss caused by myeloma, without increasing tumour burden. BMP inhibition directly reduces osteoclastogenesis, increases osteoblasts and bone formation, and suppresses bone marrow sclerostin levels. In summary we describe a novel role for the BMP pathway in myel...