Oxygen-sensing PHDs regulate bone homeostasis through the modulation of osteoprotegerin

Bone development and homeostasis are achieved through an intricate balance between osteoblastic bone formation and osteoclastic bone resorption (Zaidi 2007; Khosla et al. 2008). Mammalian bone develops through endochondral ossification, a process that requires the coordination of osteoblasts and blood vessel invasion into the cartilaginous template to trigger the formation of the primary ossification center, highlighting the importance of angiogenesis during bone formation (Kronenberg 2003; Provot and Schipani 2005; Maes 2013). Maintenance of bone homeostasis is achieved by coupling bone formation with osteoblastic bone resorption. In particular, the RANK/RANKL/osteoprotegerin (OPG) axis contributes to this process by facilitating osteoblast-mediated osteoclastogenesis (Boyle et al. 2003). Deregulation of bone homeostasis is a hallmark of pathophysiological bone disease and occurs due to imbalances in osteogenic–angiogenic coupling or between bone-forming osteoblasts and bone resorption by osteoclasts. Increased bone results in osteopetrosis, whereas osteoporosis is associated with bone loss (Teitelbaum and Ross 2003; Seeman and Delmas 2006). Elucidating the molecular mechanisms that control bone homeostasis within the bone microenvironment is needed in order to identify novel therapeutic strategies to generate functional bone to treat chronic metabolic bone disorders where the options for treatment are still limited. The bone microenvironment is a particularly hypoxic microenvironment. Most adult tissues are well vascularized, allowing for the promotion of oxygen transport with normal median interstitial oxygen tension (pO2) values ranging from 3% to 9% (Vaupel et al. 1989). In contrast, pO2 levels in the bone range from <1% in hypoxic regions up to 6% in sinusoidal cavities (Mohyeldin et al. 2010). Low oxygen tensions in the bone are likely a result of low oxygen levels in sinusoidal blood and high levels of oxygen consumption by hematopoietic cells. Blood flow in the bone travels from the central artery into arterioles, capillaries, and finally sinusoids, accompanied by a progressive drop in blood velocity and oxygenation. It has been estimated that the blood flow rate in sinusoids is approximately 1/10 to 1/20 of that found in arterioles. In addition to low blood flow and oxygen tension within sinusoids, mathematical models predict that a layer of three myeloid progenitors is sufficient to consume most oxygen provided by a nearby sinusoid (Chow et al. 2001; Winkler et al. 2010, Rankin et al. 2011). Immunohistochemical staining and microcomputer tomography (μCT) imaging of bone demonstrate that blood vessels within the adult bone and bone marrow display large structural and spatial distributional heterogeneity (Kusumbe et al. 2014; Regan et al. 2014; Spencer et al. 2014). Hence, this tissue displays vascular heterogeneity resulting in regionally defined areas of low oxygen tension or hypoxia. Despite the fact that the bone resides in a hypoxic microenvironment, the contribution of this oxygen gradient and the role of the cellular oxygen sensors in regulating bone homeostasis remain poorly understood. Prolyl hydroxylase (PHD) enzymes (PHD1, PHD2, and PHD3) are the primary cellular oxygen sensors (Semenza 2001). Most notably, PHD enzymes use oxygen as a molecular substrate to regulate the protein stability of the hypoxia-inducible transcription factors (HIF-1α and HIF-2α) in an oxygen-dependent manner (Schofield and Ratcliffe 2004). At oxygen tensions >5%, the PHD enzymes regulate HIF protein stability by catalyzing the hydroxylation of HIF transcription factors on conserved proline residues, targeting them for proteosomal degradation by the von Hippel-Lindau (pVHL) ubiquitin ligase complex. When oxygen tensions fall below 5%, PHD enzyme activity is diminished, and HIF-1α and HIF-2α are stabilized and activate the expression of genes involved in diverse physiological conditions ranging from erythropoiesis to metabolism to angiogenesis (Semenza 2012). Thus, PHD enzymes provide a direct link between oxygen availability and the regulation of HIF transcriptional activity. While the PHD isoforms have nearly ubiquitous tissue expression, the tissue-specific role of each isoform in regulating HIF protein levels remains poorly defined. Here, we sought to dissect the physiologic role of oxygen-sensing PHD1, PHD2, and PHD3 enzymes in the regulation of bone homeostasis. For this purpose, we used a genetic approach to specifically inactivate Phd1, Phd2, and Phd3 in cells of the osteoblastic lineage using Cre-loxP-mediated recombination. In addition to coordinating osteoblastic–angiogenic coupling, we identified a previously unknown role for the PHD/HIF signaling pathway in the osteoblastic regulation of osteoclastogenesis through the direct regulation of OPG. Importantly, inactivation of selective PHD isoforms within the cells of the osteoblastic lineage inhibited bone loss in ovariectomized mice. Thus, molecular oxygen, through the PHD/HIF signaling pathway, plays a central role in bone homeostasis by controlling both angiogenesis and osteoclastogenesis, and therapeutic targeting of PHD enzymes can potentially be used for the treatment of bone disorders.