Regulation of osteoblast and osteoclast differentiation by c-Fos and Msx transcription factors
Bone cell differentiation and remodelling are controlled by hormones, growth factors and specific transcriptional regulators. This thesis aims to investigate the role of two transcription factors, c-Fos/AP-1 and Msx-1/2 in osteoclast and osteoblast differentiation using in vivo and in vitro approaches. c-Fos has been shown previously to be essential for osteoclast differentiation and is overexpressed in osteoclasts of Paget's disease. To investigate the role of c-Fos in osteoclasts, transgenic mice were generated where c-fos was overexpressed in osteoclasts using the TRAP promoter. Several TRAP-c-fosLTR transgenic founders were generated which developed severe bone remodelling lesions with eventual tumour formation. Histological and in situ expression studies showed abundant osteoclasts within these lesions which expressed c-Fos, in addition to the anti-apoptotic gene Bcl-2. These features are reminiscent of Pagetic osteoclasts and suggest that these mice are useful for studying bone remodelling disorders. The effects of c-Fos on osteoblasts was next investigated in vitro using a well defined inducible expression system. Stable MC3T3-E1 osteoblastic subclones expressing a tetracycline-regulatable c-fos gene demonstrated that exogenous c-Fos appeared to augment the proliferation induced by BMP-2, and inhibited BMP-2-induced alkaline phosphatase activity during differentiation. Moreover, ectopic c-Fos expression in these cells stimulated apoptosis induced by serum withdrawal and Etoposide. This apoptosis was not effectively blocked by the caspase inhibitors Z-VAD-fmk and DEVD-CHO, but was blocked by the cell cycle dependent kinase (CDK) inhibitor, Roscovitine, and ectopic Bcl-2 or p2lWAF1,CIP1,SD11 expression. These results may provide a novel link between growth control and apoptosis in osteoblasts, such that under environmental stress, c-Fos may drive cell cycle progression and render the cell susceptible to apoptosis. Finally, the regulation of Msx2 by osteotropic factors using two reporter gene constructs was analysed in osteoblastic cells. PTH showed no regulation of Msx2 expression, however, small increases were observed with BMPs. The expression of Msx1 during bone development was also analysed in tissues from Msx1-lacZ transgenic mice. LacZ expression was detected in mineralising tissues of the foetus and neonate, but no LacZ expression was observed after birth. Taken together, these data further delineate the functional roles of these transcription factors in bone development and bone disease. Importantly, these studies provide a role for c-Fos in osteoblast apoptosis and osteoclast function, and may serve as a model for c-Fos overexpression in Paget's disease.