Medicine (St Vincent's) - Theses
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ItemOsteoblastic EphrinB2 in normal and parathyroid hormone (PTH)-stimulated bone formation.( 2016)Bone remodelling renews the skeleton through cycles of bone resorption by osteoclasts and formation by osteoblasts. Two interacting members of the Eph tyrosine kinase family, EphrinB2 and EphB4, are expressed by osteoblasts and their expression of EphrinB2 is stimulated by parathyroid hormone (PTH), the only approved anabolic therapy for osteoporosis. I investigated the roles of the ephrinB2/EphB4 interaction in osteoblast lineage cells in bone remodelling and mediating anabolic actions of PTH. I began by assessing the effects of EphB4 overexpression in mouse osteoblasts in vivo and how this affected anabolic PTH. While PTH induced increases in osteoblast and osteoid parameters in both wild-type and EphB4-overexpressing mice, these changes were not significantly different between the two groups. However, we observed some evidence of a suppressed osteoclast response to PTH, and a slight augmentation of the anabolic action of PTH on bone volume in female mice only. Lack of consistent overexpression of EphB4 in these mice limited our ability to gain further conclusions. Next, I investigated the effects of pharmacological blockade of ephrinB2/EphB4 interaction in vivo in the presence and absence of anabolic PTH. A previously described pharmacologic inhibitor of this interaction, sEphB4, was used alone or in combination with anabolic PTH. In both settings, sEphB4 treatment increased osteoblast formation and mRNA levels of early osteoblast markers (Runx2, alkaline phosphatase, Collagen 1a1, and PTH receptor [PTHR1]) without causing significantly higher bone formation rate or late markers of osteoblast/osteocyte differentiation. In the presence of PTH, sEphB4 treatment significantly increased osteoclast formation and converted the anabolic effect of PTH to a catabolic effect. This effect on osteoclasts was recapitulated in vitro in co-cultures, suggesting that it is a cell-autonomous effect. This indicates a key role for the ephrinB2/EphB4 interaction within the osteoblast lineage in osteoblast differentiation and support of osteoclastogenesis. To assess the role of ephrinB2 signalling in the osteoblast lineage in normal bone formation, I analyzed bone remodelling in mice that lacked osteoblastic ephrinB2. For this purpose, an osteoblast-specific genetic deletion mouse model was used. These mice showed a delay in bone mineralisation along with lower mRNA levels of late osteoblast differentiation markers and greater levels of osteoblast and osteocyte apoptosis. Additionally, osteoblastic support of osteoclast formation was defective in these mice and in osteoblasts cultured from these mice. Finally, I assessed how lack of osteoblastic ephrinB2 signalling affected anabolic PTH effects by treating the above-mentioned mice with anabolic PTH. This experiment showed that osteoblastic ephrinB2 is required, not only for physiological bone remodelling, but also for effects of anabolic PTH in causing higher osteoblast number, surface and osteoid production. This requirement appears to be due to the control ephrinB2 may have over osteoid deposition and mineralisation in trabecular and cortical bone. I conclude that ephrinB2, through its binding partner EphB4, promotes the late stages of osteoblast maturation that are required for mineralisation, and supports osteoclast formation. These functions are essential for the full anabolic action of PTH, such that blockade of signalling events downstream of the ephrinB2:EphB4 interaction turns anabolic action of PTH into a catabolic effect. Given the current interest in targeting the ephrinB2/EphB4 interaction in antiangiogenic and anticancer therapies, my findings show that targeting EphB4 may have less skeletal side effects than blocking both sides of this interaction. Enhancing ephrinB2 signalling may be a viable strategy for conditions where more bone formation is desired such as osteoporosis, osteogenesis imperfecta, and fracture healing.
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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.