Title: The role of prolyl hydroxylases of hypoxia inducible factor in bone development, homeostasis and pathology
Other Titles: De rol van prolyl hydroxylases van hypoxie induceerbare factor in bot ontwikkeling, homeostase en pathologie
Authors: Laperre, Kjell; M0200236
Issue Date: 2-Jul-2013
Abstract: Oxygen and nutrients are critical for the survival of cells. Beyond a certain size of a tissue, simple diffusion of oxygen becomes inadequate and cells will turn into a hypoxic state. An important aspect of cell survival is the ability to adapt to this stress situation. This adaptation encompasses on the one hand stimulation of angiogenesis to restore oxygen and nutrient supply, and on the other hand changes in cell metabolism to optimize energy production and limit energy expenditure. The central mediator of this hypoxia signaling pathway is the hypoxia inducible factor (HIF), which promotes the expression of several HIF target genes that regulate the hypoxic response. HIF-a protein levels are tightly controlled by three prolyl hydroxylase domain-containing (PHD) proteins, considered as the main oxygen sensors of the cell, namely PHD1, PHD2 and PHD3. Bone and cartilage are very active metabolic tissues and therefore need a balanced supply of oxygen and nutrients. In addition, hypoxia is present in the developing growth plate, as well as in the bone marrow and osteocytes lacunae during normal bone homeostasis. Moreover, several bone pathologies including fractures are characterized by the presence of hypoxia. The importance of HIF signalling in bone development was already demonstrated by other groups. Deletion or overexpression of HIF in osteoblasts results in highly decreased or increased bone mass, respectively. Furthermore, HIF functions as a survival factor in chondrocytes as lack of HIF-1a in chondrocytes leads to massive apoptosis of the centrally localised chondrocytes. Still, the exact role of PHD proteins, their effect on HIF signaling and on cellular processes in bone is still unknown. The aim of this study was to investigate the involvement of the PHD proteins in bone development, homeostasis and pathology.In the first chapter, we analyzed the bone phenotype of Phd1 and Phd3 null mice. According to what has been reported before, both Phd1 and Phd3 null mice show no gross abnormalities and develop normally. We observed that also bone development and homeostasis was comparable in wild type and Phd1 or Phd3 null mice. It has been suggested that PHD1 and PHD3 exert a more important function in hypoxic (pathological) conditions. Therefore the role of PHD1 and PHD3 was studied in 2 models of osteoporotic bone loss that are associated with a certain degree of hypoxia, namely hind limb unloading and ovariectomy. However, loss of Phd1 or Phd3 did not rescue or aggravate bone loss induced by unloading or ovariectomy. In the second chapter, the role of PHD2 in bone development was addressed. Since Phd2 null mice die in utero before bone development starts, we genetically inactivated Phd2 in chondrocytes by crossing Phd2fl with collagen 2-Cre mice (Phd2chon-). Inactivation of Phd2 was efficient and cell-specific, and resulted in highly increased HIF-1a protein levels in the chondrocytes. Phenotypically, Phd2chon- mice were growth retarded and unexpectedly, trabecular bone volume was almost doubled. Yet, no major differences in bone formation, resorption or vascularisation were observed. Cartilage remnants were, however, still present in the trabeculae of Phd2chon- mice, suggesting that the cartilage matrix was modified and hence hampered its remodeling by osteoclasts. Mechanistically, Phd2 null chondrocytes adjusted their energy metabolism in response to the increased HIF-1a levels without jeopardizing cell viability: energy production as well as energy expenditure was reduced. Indeed, Phd2 null chondrocytes displayed a shift towards anaerobic metabolism characterized by increased glycolysis and lactate production, whereas glucose oxidation was decreased and associated with reduced mitochondrial biogenesis. This adaptation resulted in decreased oxygen consumption and reduced ATP levels. To withstand this challenge, Phd2 null chondrocytes reduced their energy expenditure by decreasing proliferation and activating the unfolded protein response. Protein synthesis and in particular collagen synthesis was reduced. Posttranslational collagen processing was, however, promoted, evidenced by increased mRNA levels of the collagen folding enzymes protein disulfide isomerase and lysyl oxidase, and the presence of abundant collagen crosslinks. The collagen network in Phd2 null growth plate cartilage was denser and cartilage matrix mineralization was enhanced, changes that may render the collagen matrix less susceptible to degradation and lead to the formation of cartilage remnants.In conclusion, we showed that single deletion of Phd1 or Phd3 does not hamper normal bone development and homeostasis, nor influences trabecular bone loss induced by ovariectomy or hind limb unloading. On the other hand, deletion of Phd2 in growth plate chondrocytes lowers cellular energy metabolism and activates the unfolded protein response. Hence crosslinked collagens accumulate in the cartilage matrix and promote matrix mineralization but preclude remodeling, which finally results in increased bone mass.
Table of Contents: Abbreviations

Chapter 1: Introduction
1.1 Hypoxia signaling cascade
1.1.1 Hypoxia inducible factor (HIF) HIF family HIF function Regulation of HIF
1.1.2 Prolyl hydroxylase domain-containing proteins (PHDs) PHD family PHD function Regulation of PHD
1.1.3 Hypoxia-induced cellular adaptations Angiogenesis Energy metabolism Protein synthesis Ion transport Proliferation
1.2 Skeletal development
1.2.1 Bone development Intramembranous ossification Endochondral ossification
1.2.2 Bone homeostasis Bone formation Bone resorption
1.2.3 Extracellular matrix production
1.3 Hypoxia signaling and bone
1.3.1 HIF signaling in bone development
1.3.2 HIF signaling in bone pathology

Chapter 2: Aims and objectives of the study

Chapter 3: Materials and methods
3.1 Animal models
3.1.1 Transgenic mice
3.1.2 Genotyping
3.1.3 Mouse models of osteoporotic bone loss Skeletal unloading Ovariectomy
3.2 Cell cultures
3.2.1 Primary chondrocyte cultures Isolation of primary chondrocytes
3.2.2 Primary osteoblast cultures
3.2.3 Primary bone marrow stromal cell cultures
3.2.4 Primary osteoclast cultures
3.3 Gene and protein expression analysis
3.3.1 Real-time quantitative RT-PCR (qRT-PCR)
3.3.2 In situ hybridization
3.3.3 Protein isolation and western blotting
3.4 Metabolic assays
3.4.1 Enzyme-linked immunosorbent assay (ELISA)
3.4.2 Metabolic profiling
3.4.3 Mitochondrial content assessment
3.4.4 Lysosome detection
3.4.5 Intracellular calcium measurements
3.4.6 Protein translation quantification
3.5 Histological and morphometric analysis
3.5.1 Optical microscopy Pre-sacrifice treatments Embedding and sectioning Histochemical staining Immunohistochemical staining Bone histomorphometry
3.5.2 Transmission electron microscopy (TEM)
3.5.3 Fourier-transformed infra red microscopy (FTIR)
3.5.4 Peripheral quantitative computed tomography (pQCT)
3.5.5 Micro-computed tomography (micro-CT)
3.5.6 Whole-body dual-energy X-ray absorptiometry (DEXA)
3.6 Amino acid composition analysis
3.7 Cartilage collagen cross-link analysis
3.8 Serum and urine biochemistry
3.9 Statistical analysis

Chapter 4: PHD1 and PHD3 are redundant in bone development, homeostasis and pathology
4.1 Introduction
4.2 Results
4.2.1 Phd1-/- and Phd3-/- mice display a normal growth and body composition
4.2.2 Phd1-/- and Phd3-/- mice show normal bone structure
4.2.3 PHD1 and PHD3 do not influence ovariectomy or unloading-induced bone loss
4.2.4 Gene expression analysis of Phd1-/- and Phd3-/- primary bone cells
4.3 Discussion

Chapter 5: Insufficient mitochondrial respiration in Phd2-deficient growth plate chondrocytes causes skeletal dysplasia
5.1 Introduction
5.2 Results
5.2.1 PHD2 in chondrocytes regulates bone growth
5.2.2 Cartilage remnants lead to increased trabecular bone volume in Phd2chon- mice
5.2.3 Phd2 deficient chondrocytes are energy deficient
5.2.4 Mitochondrial respiration and biogenesis are decreased in Phd2 null chondrocytes
5.2.5 PHD2 regulates proliferation and Ca2+ homeostasis in chondrocytes
5.2.6 Deletion of Phd2 in chondrocytes results in activation of the unfolded protein response
5.2.7 Cartilage matrix properties are changed by Phd2 deletion
5.3 Discussion

Chapter 6: General conclusion and perspectives
6.1 PHD1 and PHD3 are redundant in bone development, homeostasis and pathology
6.2 PHD2 in chondrocytes infuences bone mass by regulating cartilage collagen processing
6.3 Clinical perspectives




Curriculum Vitae
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Clinical and Experimental Endocrinology
Laboratory of Angiogenesis and Vascular Metabolism (Vesalius Research Center) (+)
Molecular and Vascular Biology

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