Richa Khatri

The HIF Signaling Pathway in Osteoblasts Directly Modulates Erythropoiesis through the Production of EPO

Osteoblasts are an important component of the hematopoietic microenvironment in bone. However, the mechanisms by which osteoblasts control hematopoiesis remain unknown. We show that augmented HIF signaling in osteoprogenitors results in HSC niche expansion associated with selective expansion of the erythroid lineage. Increased red blood cell production occurred in an EPO-dependent manner with increased EPO expression in bone and suppressed EPO expression in the kidney. In contrast, inactivation of HIF in osteoprogenitors reduced EPO expression in bone. Importantly, augmented HIF activity in osteoprogenitors protected mice from stress-induced anemia. Pharmacologic or genetic inhibition of prolyl hydroxylases1/2/3 in osteoprogenitors elevated EPO expression in bone and increased hematocrit. These data reveal an unexpected role for osteoblasts in the production of EPO and modulation of erythropoiesis. Furthermore, these studies demonstrate a molecular role for osteoblastic PHD/VHL/HIF signaling that can be targeted to elevate both HSCs and erythroid progenitors in the local hematopoietic microenvironment.

Role of hypoxia-inducible factor-1α in angiogenic―osteogenic coupling

Angiogenesis and osteogenesis are tightly coupled during bone development and regeneration. The vasculature supplies oxygen to developing and regenerating bone and also delivers critical signals to the stroma that stimulate mesenchymal cell specification to promote bone formation. Recent studies suggest that the hypoxia-inducible factors (HIFs) are required for the initiation of the angiogenic–osteogenic cascade. Genetic manipulation of individual components of the HIF/vascular endothelial growth factor (VEGF) pathway in mice has provided clues to how coupling is achieved. In this article, we review the current understanding of the cellular and molecular mechanisms responsible for angiogenic–osteogenic coupling. We also briefly discuss the therapeutic manipulation of HIF and VEGF in skeletal repair. Such discoveries suggest promising approaches for the development of novel therapies to improve bone accretion and repair.

VEGF-independent cell-autonomous functions of HIF-1α regulating oxygen consumption in fetal cartilage are critical for chondrocyte survival

Fetal growth plate cartilage is nonvascularized, and chondrocytes largely develop in hypoxic conditions. We previously found that mice lacking the hypoxia‐inducible transcription factor HIF‐1α in cartilage show massive death of centrally located, hypoxic chondrocytes. A similar phenotype was observed in mice with genetic ablation of either all or specifically the diffusible isoforms of vascular endothelial growth factor (VEGF), a prime angiogenic target of HIF‐1α. Here, we assessed whether VEGF is a critical downstream component of the HIF‐1α–dependent survival pathway in chondrocytes. We used a genetic approach to conditionally overexpress VEGF164 in chondrocytes lacking HIF‐1α, evaluating potential rescuing effects. The effectiveness of the strategy was validated by showing that transgenic expression of VEGF164 in Col2‐Cre;VEGFf/f mice stimulated angiogenesis in the perichondrium, fully corrected the excessive hypoxia of VEGF‐deficient chondrocytes, and completely prevented chondrocyte death. Yet, similarly crossed double‐mutant embryos lacking HIF‐1α and overexpressing VEGF164 in the growth plate cartilage still displayed a central cell death phenotype, albeit slightly delayed and less severe compared with mice exclusively lacking HIF‐1α. Transgenic VEGF164 induced massive angiogenesis in the perichondrium, yet this only partially relieved the aberrant hypoxia present in HIF‐1α–deficient cartilage and thereby likely inflicted only a partial rescue effect. In fact, excessive hypoxia and failure to upregulate phosphoglycerate‐kinase 1 (PGK1), a key enzyme of anaerobic glycolytic metabolism, were among the earliest manifestations of HIF‐1α deficiency in cartilaginous bone templates, and reduced PGK1 expression was irrespective of transgenic VEGF164. These findings suggest that HIF‐1α activates VEGF‐independent cell‐autonomous mechanisms to sustain oxygen levels in the challenged avascular cartilage by reducing oxygen consumption. Hence, regulation of the metabolic pathways by HIF‐1α and VEGF‐dependent regulation of angiogenesis coordinately act to maintain physiological cartilage oxygenation. We conclude that VEGF and HIF‐1α are critical preservers of chondrocyte survival by ensuring an adequate balance between availability and handling of oxygen in developing growth cartilage.

Lack of HIF-2α in limb bud mesenchyme causes a modest and transient delay of endochondral bone development.

Lack of HIF-2α in limb bud mesenchyme causes a modest and transient delay of endochondral bone development

Osteoprotegerin abrogated cortical porosity and bone marrow fibrosis in a mouse model of constitutive activation of the PTH/PTHrP receptor.

Intracortical porosities and marrow fibrosis are hallmarks of hyperparathyroidism and are present in bones of transgenic mice expressing constitutively active parathyroid hormone/parathyroid hormone-related protein receptors (PPR*Tg). Cortical porosity is the result of osteoclast activity; however, the etiology of marrow fibrosis is poorly understood. While osteoclast numbers and activity are regulated by osteoprotegerin (OPG), bisphosphonates suppress osteoclast activity but not osteoclast numbers. We therefore used OPG and bisphosphonates to evaluate the extent to which osteoclasts, as opposed to bone resorption, regulate marrow fibrosis in PPR*Tg mice after treatment of animals with vehicle, OPG, alendronate, or zoledronate. All three agents similarly increased trabecular bone volume in both PPR*Tg and control mice, suggesting that trabecular bone resorption was comparably suppressed by these agents. However, the number of trabecular osteoclasts was greatly decreased by OPG but not by either alendronate or zoledronate. Furthermore, intracortical porosity and marrow fibrosis were virtually abolished by OPG treatment, whereas alendronate and zoledronate only partially reduced these two parameters. The greater reductions in cortical porosity and increments in cortical bone mineral density with OPG in PPR*Tg mice were associated with greater improvements in bone strength. The differential effect of OPG versus bisphosphonates on marrow fibrosis, despite similar effects on trabecular bone volume, suggests that marrow fibrosis was related not only to bone resorption but also to the presence of osteoclasts.

Loss of VHL in mesenchymal progenitors of the limb bud alters multiple steps of endochondral bone development

Adaptation to low oxygen tension (hypoxia) is a critical event during development. The transcription factors Hypoxia Inducible Factor-1α (HIF-1α) and HIF-2α are essential mediators of the homeostatic responses that allow hypoxic cells to survive and differentiate. Von Hippel-Lindau protein (VHL) is the E3 ubiquitin ligase that targets HIFs to the proteasome for degradation in normoxia. We have previously demonstrated that the transcription factor HIF-1α is essential for survival and differentiation of growth plate chondrocytes, whereas HIF-2α is not necessary for fetal growth plate development. We have also shown that VHL is important for endochondral bone development, since loss of VHL in chondrocytes causes severe dwarfism. In this study, in order to expand our understanding of the role of VHL in chondrogenesis, we conditionally deleted VHL in mesenchymal progenitors of the limb bud, i.e. in cells not yet committed to the chondrocyte lineage. Deficiency of VHL in limb bud mesenchyme does not alter the timely differentiation of mesenchymal cells into chondrocytes. However, it causes structural collapse of the cartilaginous growth plate as a result of impaired proliferation, delayed terminal differentiation, and ectopic death of chondrocytes. This phenotype is associated to delayed replacement of cartilage by bone. Notably, loss of HIF-2α fully rescues the late formation of the bone marrow cavity in VHL mutant mice, though it does not affect any other detectable abnormality of the VHL mutant growth plates. Our findings demonstrate that VHL regulates bone morphogenesis as its loss considerably alters size, shape and overall development of the skeletal elements.

About the importance of being desulfated

Sulfated proteoglycans have important structural and signaling functions in the growth plate. In the October 1, 2008, issue of Genes & Development, Settembre and colleagues (2645-2650) report that lack of SUMF1, a crucial enzyme in the activation of sulfatases, causes a severe chondrodysplasia by augmenting fibroblast growth factor signaling and by hampering the autophagic process, which the investigators show is constitutively on in chondrocytes. The findings highlight the essential role of desulfation in cartilage biology and organogenesis.

Fibrosis and hypoxia-inducible factor-1α-dependent tumors of the soft tissue on loss of von Hippel-Lindau in mesenchymal progenitors

The hypoxia-inducible factor (Hif)-1α (Hif-1α) and Hif-2α (Epas1) have a critical role in both normal development and cancer. von Hippel Lindau (Vhl) protein, encoded by a tumor suppressor gene, is an E3 ubiquitin ligase that targets Hif-1α and Epas1 to the proteasome for degradation. To better understand the role of Vhl in the biology of mesenchymal cells, we analyzed mutant mice lacking Vhl in mesenchymal progenitors that give rise to the soft tissues that form and surround synovial joints. Loss of Vhl in mesenchymal progenitors of the limb bud caused severe fibrosis of the synovial joints and formation of aggressive masses with histologic features of mesenchymal tumors. Hif-1α and its downstream target connective tissue growth factor were necessary for the development of these tumors, which conversely still developed in the absence of Epas1, but at lower frequency. Human tumors of the soft tissue are a very complex and heterogeneous group of neoplasias. Our novel findings in genetically altered mice suggest that activation of the HIF signaling pathway could be an important pathogenetic event in the development and progression of at least a subset of these tumors.

The Role of Hypoxia-Induced Factors

The definition of “physiologically” normoxic conditions for either embryonic or adult cells varies significantly. The vast majority of adult normal tissues function at oxygen (O2) levels between 2 and 9%, with ambient air at 21% O2 [135]. Bone marrow, cartilage, kidney medulla, and thymus, on the other hand, can exist at 1% O2 or lower [135]. In general, when O2 tension reaches below 2%, this condition is considered to be moderate hypoxia. When O2 tension goes below 0.5%, hypoxia is considered severe. Hypoxia is not only a pathophysiological component of many human disorders, including cancer and ischemic diseases, but is also a critical factor in fetal development and differentiation [21, 39, 82]. Before the circulatory system is established, mammalian development proceeds in a relatively low O2 environment of about 3% [100, 120]. Moreover, studies that have utilized small-molecule hypoxia markers have shown the existence of specific regions of moderate to severe hypoxia in the developing embryos [21, 82].