Growth of the craniofacial complex, the skull and the mandibular condyle is dependent on the activities of chondrocytes contained within the endochondral growth plate. Maturation of cells in the growth plate is accompanied by stage specific changes in energy generation. Recent studies show that metabolic control is mediated by Hypoxia Inducible Factor (HIF), a transcription factor that responds to changes in the local oxygen tension. It is now known that HIF activity is controlled by a group of prolyl hydroxylases (PHDs) which serve as oxygen sensors. Based on these observations, we hypothesize that in the growth plate, PHD controlled activation of HIF transcriptional activity is required for chondrocyte maturation, terminal differentiation and the induction of apoptosis. Investigations described in this proposal are aimed at testing this novel hypothesis that brings together current ideas concerning chondrocyte metabolism, the impact of the local environment, the development of the terminal differentiated state and the mechanism of cell death. Thus, in Specific Aim 1, we measure the expression and distribution of the PHDs in chondrocytes undergoing terminal differentiation and ascertain how changes in PHD expression modulate HIF expression, HIF transcriptional activity and development of the terminally differentiated state. We then relate PHD expression to development of the hypertrophic state in the mouse epiphyseal growth plate.
In Aim 2, we determine the expression and distribution of HIF-la in chondrocytes undergoing terminal differentiation and ascertain how HIF expression modulates the expression of each of the PHD isoforms in the mouse growth plate.
In Aim 3, we explore how modulation of the oxemic status regulates terminal differentiation and the induction of chondrocyte apoptosis;we relate HIF-PHD expression to chondrocyte apoptosis/survival in the mouse growth plate. Finally, in the last Specific Aim, we engineer transgenic animals that conditionally over-express the PHDs. We use this model to assess the impact of PHD expression on the development of the murine epiphyseal growth plate. We relate over-expression to HIF-1 expression, chondrocyte metabolism and the activity of the apoptosis/survival pathways. Outcomes from this investigation should help define the linkage between PHDs and downstream events linked to the chondrocyte maturation process.
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