As we age, skeletal integrity becomes compromised due to a decrease in bone mineral density. Bone formation is mediated by osteoblast (OB) cells which originate from mesenchymal stem cells (MSC). MSCs, a rare stem cell population within the bone marrow, possess self-renewal and multi-lineage differentiation potential. In aging or diseased states, such as osteoporosis, there is a reduction in osteoblastic differentiation potential of MSCs in favour of differentiation toward fat storing adipocytes (AdC). Several signalling pathways have been shown to regulate osteogenic commitment of MSCs as well as the subsequent processes of osteoblast differentiation and skeletal formation. The mammalian target of rapamycin complex 1 (mTORC1) has been implicated as a master regulator of cell metabolism that integrates signals which control MSC commitment and OB function. However, the direct role of this complex in these functions remains to be determined. To definitively address the role of mTORC1 in MSC fate determination, OB differentiation and bone accrual, this project utilised the Cre-loxP system of targeted transgenesis, which enabled the tissue-specific and temporal inactivation of Rptor, the gene encoding for the mTORC1 complex-specific protein raptor. In this study Rptor-deficient MSCs cultured under osteogenic and adipogenicinductive conditions displayed a reduced capacity to form lipid-laden AdCs and an increased capacity to form a mineralised matrix. Consistent with the increased osteogenic differentiation, deletion of Rptor in MSC resulted in an up-regulation in the expression of the osteogenic growth factor BMP2, a known inducer of RUNX2 expression, OB maturation and mineral formation. To examine the role of raptor in skeletal development, Rptor was deleted in preosteoblastic cells marked by their expression of the osterix (Osx) gene, by crossing Rptorfl/fl mice with Osx-cre transgenic mice. Deletion of Rptor lead to a reduction in limb length at birth and post-natally and was associated with smaller epiphyseal growth plates. Deletion of Rptor caused a marked reduction in pre- and post-natal bone acquisition in both intramembranous and endochondral ossification leading to skeletal fragility. The decrease in bone acquisition was not due to a reduction in OB numbers but a reduction in OB function. In vitro, primary OBs from knockout animals failed to respond to extracellular factors that promote bone formation including insulin and BMP2 and assessment of bone development markers in Rptor knockout OBs revealed a transcriptional profile consistent with an immature OB phenotype suggesting that OB differentiation was hindered early in osteogenic development. These findings demonstrate that mTORC1 plays an important role in skeletal development by controlling OB differentiation and hence function. Taken together, these studies show that mTORC1 plays an important role in MSC fate determination and bone accrual. Notably, deletion of raptor in pre-osteoblasts blocked osteoblast differentiation causing defective intramembranous and endochondral ossification resulting in a low bone mass phenotype and skeletal fragility.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 201
