Nrf1 Molecular Pathway, Vitamin C Deficiency, and Spontaneous Fractures

描述（由申请人提供）： 骨质疏松症是美国军队和普通人群的一个重要健康问题，造成了巨大的经济负担。因此，成功地减少骨质疏松性骨折的数量将改善健康状况，并节省大量的医疗保健费用。为了鉴定可能在骨质疏松性骨折发生中起重要作用的候选基因，我们正在研究一种缺失古洛糖酸内酯氧化酶基因的突变小鼠，它参与合成抗坏血酸（AA），抗坏血酸（AA）在5- 10岁的极早年龄时发生自发性骨折（sfx）。AA在骨骼生物学中的重要性是由以下事实强调的：只有少数遗传异常在年轻时导致sfx，并且越来越多的流行病学证据表明，AA摄入量低的个体具有降低的骨量、更大的骨丢失率和增加的骨折。由于AA信号通路中任何分子的遗传缺陷都可能增加骨质疏松性骨折的风险，因此充分阐明AA信号通路的组成部分及其在骨中的作用至关重要。在我们的研究中，在目前的资助期内，我们已经确定，AA是必不可少的表达增加osterix（Osx），一个主成骨转录因子，在分化成骨细胞（OB）。我们的建议将解决一个悬而未决的关键问题，AA，抗氧化剂，如何调节核因子的转录活性。为此，我们获得了脯氨酰羟化酶结构域蛋白（PHD）-2参与介导OB中AA效应的新的令人兴奋的初步数据。基于已发表的数据，即p53阻断OB分化、Osx表达和骨发育，以及我们的初步数据，即AA通过PHD 2依赖性机制降低p53蛋白水平，我们将通过检验3种假设来检验涉及PHD 2和p53的AA调节Osx表达的模型。为了检验假设1，即PHD 2通过以不依赖于HIF-11的方式调节Osx表达，参与介导AA对OB分化和骨形成（BF）的影响，我们将：1）确定OB产生的PHD 2参与介导AA对OB分化的影响通过腺病毒（Ad）-Cre介导的对PHD 2的破坏在floxed OB中; 2）确定OB分化过程中AA诱导的Osx表达由PHD 2介导，不依赖于HIF 11; c）通过使用Cre/loxP技术条件性破坏PHD 2来确定PHD 2在BF调节中的体内作用;和4）通过产生AA缺陷的PHD 2条件性KO小鼠，确认PHD 2是AA拯救sfx小鼠中BF缺陷所需的。为了检验假设2，即AA激活PHD 2导致p53的脯氨酰羟基化以调节其被蛋白体降解，我们将：1）确定AA通过PHD 2调节p53的脯氨酰羟基化; 2）确定PHD 2介导的p53的脯氨酰羟基化是否经历泛素介导的蛋白体降解;和3）通过突变p53中潜在的羟基化位点并确定突变的p53是否对蛋白酶体降解具有抗性以及在抑制Osx表达方面是否比WT更有效来鉴定脯氨酰羟基化位点。为了验证假设3，即p53与Osx启动子的抗氧化反应元件（antioxidant response element，ARE）相互作用并与bZIP转录因子竞争抑制Osx转录，我们将：1）使用来自PHD 2条件性KO和WT小鼠的OB进行EMSA和ChIP分析，证实p53与Osx基因启动子的ARE结合并竞争已知与ARE相互作用的bZIP转录因子家族的成员; 2）突变Osx近端启动子中的战神，并评估p53对OB中启动子活性的影响。我们证实了PHD 2通过p53依赖性和HIF 11非依赖性机制介导AA对Osx表达和OB分化的影响，这可能导致开发新的药物靶点来增加OB分化和BF。

DESCRIPTION (provided by applicant): Osteoporosis, a significant health problem in the military as well as general population in the U.S., poses a significant financial burden. Therefore, successful efforts to reduce the number of osteoporotic fractures would lead to improved health as well as considerable savings in health care costs. To identify candidate genes that may be important in the development of osteoporotic fractures, we are studying a mutant mouse with deletion of gulunolactone oxidase gene, which is involved in the synthesis of ascorbic acid (AA) that develops spontaneous fractures (sfx) at the very early age of 5-7 wks. The importance of AA in skeletal biology is underscored by the fact only a select few genetic abnormalities lead to sfx at a young age and the accumulating epidemiological evidence that individuals with low AA intake have reduced bone mass, greater rate of bone loss and increased fractures. Since genetic defects in any of the molecules involved in AA signaling pathway could increase the risk for osteoprotoic fractures, it is important to fully elucidate the components of AA signaling pathway and their role in bone. In our studies during the current funding period, we have established that AA is essential for increased expression of osterix (Osx), a master osteogenic transcription factor, during differentiation of osteoblasts (OBs). Our proposal will address an unanswered key question of how AA, an antioxidant, modulates transcriptional activity of nuclear factors. To this end, we have new exciting preliminary data for the involvement of prolyl hydroxylase domain protein (PHD)-2 in mediating AA effects in OBs. Based on the published data that p53 blocks OB differentiation, Osx expression and bone development and our preliminary data that AA decreases p53 protein levels via PHD2-dependent mechanism, we will test a model for AA regulation of Osx expression involving PHD2 and p53 by examining 3 hypotheses. To test hypothesis 1, that PHD2 is involved in mediating AA effects on OB differentiation and bone formation (BF) via regulating Osx expression in a HIF-11-independent manner, we will: 1) Establish that OB produced PHD2 is involved in mediating AA effects on OB differentiation by Adenoviral (Ad)-Cre mediated disruption of PHD2 in floxed OBs; 2) Establish that AA-induced Osx expression during OB differentiation is mediated by PHD2, independent of HIF11; c) Ascertain in vivo role of PHD2 in the regulation of BF by conditional disruption of PHD2 using Cre/loxP technology; and 4) Confirm that PHD2 is required for AA to rescue BF deficiency in sfx mice by generating PHD2 conditional KO mice that are AA-deficient. To test hypothesis 2, that AA activation of PHD2 results in prolyl hydroxylation of p53 to regulate its degradation by proteosomes, we will: 1) Ascertain that AA regulates prolyl hydroxylation of p53 via PHD2; 2) Establish if PHD2 mediated prolyl hydroxylation of p53 is subjected to ubiquitin-mediated proteosomal degradation; and 3) Identify sites of prolyl hydroxylation by mutating potential hydroxylation sites in p53 and determining if mutated p53 is resistant to proteosomal degradation and more potent than WT in inhibiting Osx expression. To test hypothesis 3, that p53 interacts with antioxidant response element (ARE) of Osx promoter and competes with bZIP transcription factors to suppress Osx transctiption, we will: 1) Perform EMSA and ChIP assays using OBs-derived from PHD2 conditional KO and WT mice and confirm that p53 binds to ARE of Osx gene promoter and competes members of bZIP family of transcription factors known to interact with ARE; and 2) Mutate putative AREs within the proximal promoter of Osx and evaluate p53's effect on promoter activity in OBs. Our confirmation of the hypothesis that PHD2 mediates AA effects on Osx expression and OB differentiation via p53-dependent and HIF11-independent mechanism could lead to the development of new drug targets to increase OB differentiation and BF.