Within the epiphysial growth plate, activated chondrocytes undergo a series of temporal and spatial changes that lead to hypertrophy and development of the terminally differentiated phenotype. Numerous studies have shown that this cascade of events is accompanied by a switch in energy generation ranging from oxidative metabolism in the proliferative zone to anaerobic glycolysis in the hypertrophic region. This curious shift in cellular energetics probably reflects an adaptive response to the local oxygen supply. The cellular oxygen sensing mechanism whose effector molecule is the transcription factor HIF has recently been shown to involve a family of prolyl hydroxylases (PHDs), whose oxygen dependent enzymatic activity controls the activity of HIF. The importance of HIF-1 for this adaptive response is anchored by the fact that HIF-1 positively regulates glycolytic gene expression and furthermore by the presence of high levels of apoptotic cells in the proliferative zone of the growth plates in conditional HIF-la knock-out mice. We hypothesize that the oxygen gradient in the developing growth plate is sensed along the PHD-HIF axis, whose modulation is required for the metabolic adaptation during the ordered maturation process. We address this through two Specific Aims.
In Specific Aim 1 we assess PHD and HIF protein and gene expression in epiphyseal chondrocytes. We then relate expression of these indicators of oxidative metabolism to chondrocyte proliferation, hypertrophy and terminal differentiation.
In Specific Aim 2 we examine how changes in PHD expression modulates HIF expression, transcriptional activity and chondrocyte maturation and determine how changes in PHD expression modifies the response of cells to alterations in oxygen tension.
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