Medicine (St Vincent's) - Theses
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ItemRetinoic acid receptor signalling in bone and microenvironment regulation of haematopoiesis( 2016)Deficiency and high levels of vitamin A are associated with increased fracture risk and poor bone health. Derivatives of vitamin A, termed retinoids, signal through nuclear retinoic acid receptors (RARs) to regulate gene transcription. Our group previously showed that Rarg-/- mice have a microenvironment-induced haematopoietic defect, meaning RARγ signalling in non-haematopoietic cells is required for healthy blood cell production. Despite a clear role for RAR signalling in bone and extrinsic regulation of haematopoiesis our understanding of the direct roles of individual RAR subtypes in bone-forming osteoblast lineage cells requires further investigation. In this study we demonstrated that RARα and RARγ have direct regulatory roles in osteoblast differentiation by using highly selective RAR ligands. RARγ antagonist administration to early osteoprogenitor Kusa4b10 cells accelerated osteoblast differentiation, which was further enhanced by RARα antagonist treatment. In comparison, RARα and RARγ agonist administration (alone or in combination) resulted in impaired Wnt signalling and a complete block in osteoblast differentiation. The role of RARs in bone was investigated in vivo by pan-RAR antagonist administration and the use of RARγ knockout models. We found pharmacological inhibition of all RARs for 10 days inhibited radial bone growth in 8-week-old mice. Radial bone growth was also impaired in Rarg-/- mice, which also exhibited impaired longitudinal growth, reduced cortical bone thickness and trabecular bone volume. Deletion of Rarg in Prx1-Cre-targeted limb bud-derived mesenchymal stromal cells and their progeny partially recapitulated the phenotypes observed in Rarg-/- mice. Prx:RargΔ/Δ mice exhibited increased osteoclastogenesis, similarly observed in Rarg-/- mice, indicating an extrinsic role for RARγ regulation of osteoclastogenesis. In comparison, deletion in Osx-Cre-targeted osteoprogenitors had no effect on bone structure. The haematopoietic phenotype was also investigated in Prx:RargΔ/Δ mice. Prx:RargΔ/Δ male mice exhibited elevated pro-B and pre-B lymphocyte precursors in the bone marrow, implicating Prx1-targeted cells as regulators of the early B lymphopoiesis niche(s) through RARγ. To enhance our ability to understand bone marrow microenvironmental cells, we improved upon current isolation techniques of mesenchymal cells from bone using fluorescence activated cell sorting (FACS). We isolated cells from collagenase-digested bone, excluded haematopoietic (CD45+, Ter119+) and endothelial (CD31+) cells and selected for Sca-1-CD51+ cells. We then profiled and characterised four subpopulations based on PDGFRα and PDGFRβ expression, which we named A (PDGFRα+PDGFRβ-), AB (PDGFRα+PDGFRβ+), B (PDGFRα-PDGFRβ+) and double negative (DN; PDGFRα-PDGFRβ-). These populations had unique and overlapping qualities. Populations AB and B exhibited characteristics of cells implicated in early B lymphocyte niches. Preliminary experiments using this technique were then performed to study the haematopoietic defect in Prx:RargΔ/Δ and Rarg-/- mice. Collectively, these findings help to explain why vitamin A deficiency (as well as excess) is linked with poor outcomes for skeletal health. Furthermore, we show RARγ expression in Prx1-targeted cells and their progeny is involved in extrinsic regulation of osteoclastogenesis and B lymphopoiesis. This emphasises the importance of the bone marrow microenvironment in bone turnover and haematopoiesis.
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ItemMouse models of human osteosarcoma( 2015)Li-Fraumeni (P53 mutation), Retinoblastoma (RB mutation) and Rothmund Thomson Syndrome (RECQL4 mutation) are three human familial cancer syndromes that present with the strongest association to osteosarcoma (OS). While sporadic OS patients possess mutations in members of the p53 and Rb pathways, not much is known about the role of RECQL4 in benign and malignant skeletal development. With the advent and characterisation of genetically modified mouse OS models, this provides an opportunity to recapitulate some aspects of human disease. Hence, my thesis aims to characterise a novel transgenic mouse OS model that is based on the shRNA-mediated suppression of p53, and to elucidate the effects of Recql4 deletion on bone biology and OS development. My original hypotheses were that the in vivo expression of shRNA against p53 can be used to model OS, and that Recql4 plays a vital role in normal bone development and OS biology. The first hypothesis was supported by the spontaneous development of OS in mice with suppressed p53 transcript levels in osteoblasts. The primary tumours were predominantly situated in the long bones, while these mice presented with metastasis more frequently than its p53 deletion counterpart. The OS phenotype of the shRNA model was determined to be representative of the osteoblastic OS subtype, which is the most commonly diagnosed form of OS in humans. This study represents the first characterisation of a mouse model of osteoblastic OS. The second hypothesis was tested with mouse genetic models to study the loss of Recql4 and its effects on normal bone development and OS biology. Proliferative and maturation defects were seen in osteoblastic cells that lacked Recql4, which translated into reduced trabecular and cortical bone mass in vivo. While mice with the osteoblastic loss of Recql4 did not develop OS, the concurrent deletion of p53 and Recql4 led to a delayed onset of malignant disease compared to its p53-null counterpart. Also, the lack of differences in OS tumour features was attributed to the selection pressure that prevented the total deletion of Recql4. These findings highlight the importance of Recql4 in osteoblast development and OS initiation. Taken together, the work presented in my thesis has helped to improve our understanding of OS biology, as the characterisation of murine OS models in this thesis will be useful in reflecting the disease aspects and diversity seen in human OS patients.
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ItemThe role of AMPK in bone and cholesterol metabolism( 2012)The central focus of this thesis explores the biology of AMP-activated protein kinase (AMPK), an important whole-body energy regulator, firstly in bone metabolism, and secondly in the regulation of cholesterol metabolism. AMPK is a ubiquitously-expressed αβγ heterotrimeric enzyme. Since AMPK plays important roles in modulating metabolism in response to diet and exercise, both of which influence bone mass, the influence of AMPK on bone mass was investigated in mice. In the first part, I will present data that the non-specific AMPK activator, AICAR, enhanced the formation of osteoclasts (bone-resorbing cells) in vitro and caused bone loss in vivo. However, the in vitro effects were shown to be AMPK-independent. Next, I will present genetic evidence that AMPK is important for the maintenance of normal bone mass, as germline deletion of either AMPK β1 or β2 subunit isoforms resulted in reduced trabecular bone density and mass, but without affecting osteoblast or osteoclast numbers. In Chapter 6, I focussed on one of the substrates of AMPK named 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), which catalyses the rate limiting step in the mevalonate pathway. Mevalonate is essential for the synthesis of important bioactive sterols (e.g. cholesterol) and non-sterol isoprenoids, which are important for processes such as protein prenylation. Phosphorylation of residue S871 of HMGR by AMPK inhibits its activity, presumably to conserve cellular energy by shutting down cholesterol synthesis. To study the physiological importance of this phosphorylation control of HMGR by AMPK, our laboratory generated a knock-in mutation in mice by homologous recombination and Cre-mediated excision to introduce a modified exon 20 to encode Alanine at residue 871. The data suggests that lack of regulation of HMGR by AMPK in mice did not lead to any overt physical phenotypic differences or compensatory up- or down-regulation in the expression of other mevalonate pathway genes. However, these mice displayed slightly altered lipid profiles in the brain, liver and plasma. The lack of a more prominent phenotype may reflect the importance of the mevalonate pathway and its multivalent backup regulation.