HIF Prolyl Hydroxylases and Chondrocyte Maturation

描述(申请人提供)：在骨痂生长板内，激活的软骨细胞经历一系列的时间和空间变化，导致肥大和终末分化表型的发展。大量研究表明，伴随着这一系列事件的是能量生成的转换，从增殖区的氧化代谢到肥大区的厌氧糖酵解。细胞能量学的这种奇怪的变化可能反映了对当地氧气供应的适应性反应。近年来发现，以转录因子HIF为效应分子的细胞氧感应机制涉及一类依赖氧的羟化酶(PhDS)，其氧依赖的酶活性控制HIF的活性。HIF-1对这种适应性反应的重要性是因为HIF-1正向调节糖酵解基因的表达，而且在条件性HIF-1a基因敲除小鼠的生长板增殖区存在高水平的凋亡细胞。我们假设发育中的生长板中的氧梯度是沿着PhD-HIF轴感应的，它的调节是有序成熟过程中代谢适应所必需的。我们通过两个具体目标来解决这个问题。在特定的目的1中，我们检测了PHD和HIF蛋白和基因在骨痂软骨细胞中的表达。然后，我们将这些氧化代谢指标的表达与软骨细胞的增殖、肥大和终末分化联系起来。在特定的目标2中，我们研究了PHD表达的变化如何调节HIF的表达、转录活性和软骨细胞的成熟，并确定PHD表达的变化如何改变细胞对氧分压变化的反应。

DESCRIPTION (provided by applicant): 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.