Osteocytic oxygen sensing controls bone mass through epigenetic regulation of sclerostin

Preservation of bone mass is crucial for healthy ageing and largely depends on adequate responses of matrix-embedded osteocytes. These cells control bone formation and resorption concurrently by secreting the WNT/-catenin antagonist sclerostin (SOST). Osteocytes reside within a low oxygen microenvironment, but whether and how oxygen sensing regulates their function remains elusive. Here, we show that conditional deletion of the oxygen sensor prolyl hydroxylase (PHD) 2 in osteocytes results in a high bone mass phenotype, which is caused by increased bone formation and decreased resorption. Mechanistically, enhanced HIF-1 signalling increases Sirtuin 1-dependent deacetylation of the Sost promoter, resulting in decreased sclerostin expression and enhanced WNT/-catenin signalling. Additionally, genetic ablation of PHD2 in osteocytes blunts osteoporotic bone loss induced by estrogen deficiency or mechanical unloading. Thus, oxygen sensing by PHD2 in osteocytes negatively regulates bone mass through epigenetic regulation of sclerostin and targeting PHD2 elicits an osteo-anabolic response in osteoporotic models.Research Foundation – Flanders (FWO: G.0A72.13, G.096414 and G0A4216N) and P.C. from long-term structural funding – Methusalem Funding by the Flemish Government

Biological Activity of CXCL8 Forms Generated by Alternative Cleavage of the Signal Peptide or by Aminopeptidase-Mediated Truncation

Posttranslational modification of chemokines is one of the mechanisms that regulate leukocyte migration during inflammation. Multiple natural NH(2)-terminally truncated forms of the major human neutrophil attractant interleukin-8 or CXCL8 have been identified. Although differential activity was reported for some CXCL8 forms, no biological data are available for others.status: publishe

Hypoxia signaling in the regulation of bone homeostasis, angiogenesis and cell metabolism: Implications for bone tissue engineering

It is well established that oxygen and nutrients are crucial for cellular functioning, including survival, proliferation and differentiation. As simple oxygen diffusion is limited to about 200 µm, cells will become hypoxic in tissues beyond a certain size. Cells have however developed mechanisms to resist tissue hypoxia and these are mediated by hypoxia inducible factors (HIFs) that are considered as the executors of the cellular response to low oxygen together with HIF prolyl hydroxylases (PHDs) that regulate HIFs in an oxygen-dependent manner. In general, activation of the HIF pathway will ultimately result in (i) stimulation of angiogenesis to resupply oxygen and (ii) adaptations in cell metabolism to ensure adequate energy generation and redox balance. Despite the remarkable healing potential of bone tissue, about 10% of all fractures results in delayed healing or non-union. Bone tissue engineering combines biological basic research and the principles of engineering in order to repair damaged bone using skeletal progenitor or stem cells and/or appropriate signaling molecules seeded on a biocompatible scaffold. The clinical impact of cell-based constructs is however limited, which urges for a better understanding of the cellular and molecular mechanisms of bone regeneration. Upon transplantation, grafted cells encounter an injured tissue that is poorly perfused, turning them in a hypoxic state. In a first part of this PhD project, we investigated the importance of endogenous HIF-1a signaling for bone regeneration. To this end, we specifically deleted the HIF­1a isoform in periosteal progenitor cells and show that activation of HIF-1a signaling in these cells is critical for bone repair by modulating angiogenic and metabolic processes. Activation of HIF‑1a is not only crucial for blood vessel invasion, by enhancing angiogenic growth factor production, but also for periosteal cell survival early after implantation, when blood vessels have not yet invaded the construct. Mechanistically, HIF-1a signaling limits oxygen consumption in hypoxia to avoid accumulation of harmful reactive oxygen species and thereby preserves redox balance, and additionally induces a switch to glycolysis to prevent energetic distress. Although activation of endogenous HIF-1a signaling prevents massive apoptosis, the majority of the grafted cells do not survive. Therefore, we preconditioned periosteal cells to the hypoxic environment of the bone defect site through inhibition of PHD2, the most important PHD isoform in skeletal cells. This strategy increased post-implantation cell survival and improved bone regeneration, an effect that was mediated by HIF-1a. The enhanced cell viability was angiogenesis-independent, but relied on simultaneous and combined changes in glutamine and glycogen metabolism. HIF­1a stabilization stimulated glutaminase-mediated glutathione synthesis, maintaining redox homeostasis at baseline and during oxidative or nutrient stress. At the same time, HIF­1a signaling increased glycogen storage, preventing an energy deficit during nutrient or oxygen deprivation. Lastly, we showed that pharmacological inhibition of PHD2 recapitulated the adaptations in glutamine and glycogen metabolism and thus the beneficial effects on cell survival, supporting further investigations to develop novel treatment modalities for bone regeneration. Throughout life, bone is being constantly remodeled through the coupled action of bone-forming osteoblasts and bone-resorbing osteoclasts, which is controlled among others by matrix-embedded osteocytes. This observation suggests that an adequate supply of oxygen and nutrients is needed to meet the high metabolic demand of these processes. Intriguingly, the bone microenvironment during development, adult life and several pathologies is characterized by low oxygen tensions and several reports have demonstrated a functional role for HIF signaling in osteoprogenitors and osteoblasts. However, the role of the hypoxia signaling pathway in osteocytes, the key regulators of bone mass during homeostasis and pathology, remains elusive. The aim of the last part of this PhD study was therefore to investigate the role of PHD2 in osteocytes, which are considered to be crucial regulators of postnatal bone mass. Transgenic mice lacking PHD2 in osteocytes displayed a high bone mass phenotype, caused by increased bone formation and decreased resorption, processes that were associated with enhanced angiogenesis. Mechanistically, stabilization of HIF-1a in PHD2-deficient osteocytes resulted in a Sirtuin 1-dependent decrease in the WNT/b-catenin inhibitor sclerostin, which stimulates osteogenesis while limiting osteoclast activity. The enhanced angiogenic response was mediated by a HIF-1a-dependent increase in vascular endothelial growth factor. Lastly, we show that genetic ablation of PHD2 was sufficient to protect mice from osteoporotic bone loss, which was potentially mediated by sustained WNT/b-catenin signaling. In this PhD thesis, we have determined the pleiotropic role of PHD2/HIF signaling during bone regeneration and homeostasis. Moreover, our data suggest that targeting PHD2 might be an interesting strategy to improve bone regeneration and, potentially, to limit bone loss during osteoporosis.status: publishe

Hypoxia, hypoxia-inducible transcription factors and oxygen-sensing prolyl hydroxylases in bone development and homeostasis

PURPOSE OF REVIEW: To summarize the role of hypoxia signaling in skeletal cells. RECENT FINDINGS: Hypoxia occurs at several stages during bone development. Skeletal cells, like chondrocytes and osteoblasts, respond to this challenge by stabilizing the hypoxia inducible transcription factor HIF, which induces the expression of angiogenic factors and promotes glycolysis. The increased delivery of oxygen and nutrients, together with metabolic adaptations, prevent chondrocyte cell death in the growth plate and promote bone formation by osteoblasts. However, excessive HIF levels have to be avoided during bone development as the resulting metabolic maladaptations cause skeletal dysplasia. Recent studies show that HIF also targets other genes to increase bone mass: it decreases osteoclastogenesis by increasing osteoprotegerin expression and represses sclerostin expression by epigenetic mechanisms, resulting in increased bone formation and decreased resorption. Moreover, increased HIF signaling in osteolineage cells promotes primary and metastatic breast tumor growth, and induces erythropoietin (EPO) production, resulting in polycythemia. Finally, HIF can directly or indirectly through increasing EPO levels, induce the expression and processing of FGF23 and may thereby affect mineral homeostasis and vitamin D metabolism. SUMMARY: HIF signaling in skeletal cells not only affects their behavior but also influences erythropoiesis and possibly mineral homeostasis.status: publishe

The skeletal vascular system - Breathing life into bone tissue

During bone development, homeostasis and repair, a dense vascular system provides oxygen and nutrients to highly anabolic skeletal cells. Characteristic for the vascular system in bone is the serial organization of two capillary systems, each typified by specific morphological and physiological features. Especially the arterial capillaries mediate the growth of the bone vascular system, serve as a niche for skeletal and hematopoietic progenitors and couple angiogenesis to osteogenesis. Endothelial cells and osteoprogenitor cells interact not only physically, but also communicate to each other by secretion of growth factors. A vital angiogenic growth factor is vascular endothelial growth factor and its expression in skeletal cells is controlled by osteogenic transcription factors and hypoxia signaling, whereas the secretion of angiocrine factors by endothelial cells is regulated by Notch signaling, blood flow and possibly hypoxia. Bone loss and impaired fracture repair are often associated with reduced and disorganized blood vessel network and therapeutic targeting of the angiogenic response may contribute to enhanced bone regeneration.status: publishe

The vasculature: a vessel for bone metastasis

Emerging evidence indicates that the interactions between tumor cells and the bone microenvironment have a crucial role in the pathogenesis of bone metastasis and that they can influence tumor cell dissemination, quiescence and tumor growth in the bone. The vasculature is known to be critical for primary tumor growth, and anti-angiogenesis drugs are approved for the treatment of certain tumor types. The role of the vasculature in bone metastasis is less well known, but recent evidence shows that blood vessels in the bone are a key component of the local microenvironment for the tumor cells and contribute to the different consecutive phases of bone metastasis. A better insight in the importance of the vasculature for bone metastasis may help develop novel treatment modalities that either slow down tumor growth or, preferably, prevent or cure bone metastasis.status: publishe

Glutamine metabolism in osteoprogenitors is required for bone mass accrual and PTH-induced bone anabolism in male mice.

Skeletal homeostasis critically depends on the proper anabolic functioning of osteolineage cells. Proliferation and matrix synthesis are highly demanding in terms of biosynthesis and bioenergetics, but the nutritional requirements that support these processes in bone-forming cells are not fully understood. Here, we show that glutamine metabolism is a major determinant of osteoprogenitor function during bone mass accrual. Genetic inactivation of the rate-limiting enzyme glutaminase 1 (GLS1) results in decreased postnatal bone mass, caused by impaired biosynthesis and cell survival. Mechanistically, we uncovered that GLS1-mediated glutamine catabolism supports nucleotide and amino acid synthesis, required for proliferation and matrix production. In addition, glutamine-derived glutathione prevents accumulation of reactive oxygen species and thereby safeguards cell viability. The pro-anabolic role of glutamine metabolism was further underscored in a model of parathyroid hormone (PTH)-induced bone formation. PTH administration increases glutamine uptake and catabolism, and GLS1 deletion fully blunts the PTH-induced osteoanabolic response. Taken together, our findings indicate that glutamine metabolism in osteoprogenitors is indispensable for bone formation. This article is protected by copyright. All rights reserved.status: Published onlin

Inhibition of the Oxygen Sensor PHD2 Enhances Tissue-Engineered Endochondral Bone Formation

Tissue engineering holds great promise for bone regenerative medicine, but clinical translation remains challenging. An important factor is the low cell survival after implantation, primarily caused by the lack of functional vasculature at the bone defect. Interestingly, bone development and repair initiate predominantly via an avascular cartilage template, indicating that chondrocytes are adapted to limited vascularization. Given these advantageous properties of chondrocytes, we questioned whether tissue-engineered cartilage intermediates implanted ectopically in mice are able to form bone, even when the volume size increases. Here, we show that endochondral ossification proceeds efficiently when implant size is limited (≤30 mm3 ), but chondrogenesis and matrix synthesis are impaired in the center of larger implants, leading to a fibrotic core. Increasing the level of angiogenic growth factors does not improve this outcome, because this strategy enhances peripheral bone formation, but disrupts the conversion of cartilage into bone in the center, resulting in a fibrotic core, even in small implants. On the other hand, activation of hypoxia signaling in cells before implantation stimulates chondrogenesis and matrix production, which culminates in enhanced bone formation throughout the entire implant. Together, our results show that induction of angiogenesis alone may lead to adverse effects during endochondral bone repair, whereas activation of hypoxia signaling represents a superior therapeutic strategy to improve endochondral bone regeneration in large tissue-engineered implants. © 2018 American Society for Bone and Mineral Research.status: publishe

Osteocytic oxygen sensing controls bone mass through epigenetic regulation of sclerostin

Preservation of bone mass is crucial for healthy ageing and largely depends on adequate responses of matrix-embedded osteocytes. These cells control bone formation and resorption concurrently by secreting the WNT/β-catenin antagonist sclerostin (SOST). Osteocytes reside within a low oxygen microenvironment, but whether and how oxygen sensing regulates their function remains elusive. Here, we show that conditional deletion of the oxygen sensor prolyl hydroxylase (PHD) 2 in osteocytes results in a high bone mass phenotype, which is caused by increased bone formation and decreased resorption. Mechanistically, enhanced HIF-1α signalling increases Sirtuin 1-dependent deacetylation of the Sost promoter, resulting in decreased sclerostin expression and enhanced WNT/β-catenin signalling. Additionally, genetic ablation of PHD2 in osteocytes blunts osteoporotic bone loss induced by oestrogen deficiency or mechanical unloading. Thus, oxygen sensing by PHD2 in osteocytes negatively regulates bone mass through epigenetic regulation of sclerostin and targeting PHD2 elicits an osteo-anabolic response in osteoporotic models.status: publishe