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民众的一个重大健康问题，构成了重大的经济负担。因此，成功地减少骨质疏松性骨折的数量将导致改善健康状况并节省相当大的医疗费用。为了确定可能在骨质疏松性骨折的发展中起重要作用的候选基因，我们正在研究一只缺失古龙内酯氧化酶基因的突变小鼠，该基因参与抗坏血酸(AA)的合成，在非常早的5-7周大时就会发生自发性骨折(SFX)。AA在骨骼生物学中的重要性被以下事实所强调：只有极少数基因异常会导致年轻时的SFX，而且越来越多的流行病学证据表明，AA摄入量低的人骨量减少，骨丢失率更高，骨折增加。由于AA信号通路中的任何一个分子的遗传缺陷都会增加骨原性骨折的风险，因此充分阐明AA信号通路的组成及其在骨中的作用是非常重要的。在我们目前资助期间的研究中，我们已经证实AA对于成骨细胞(OBS)分化过程中主要成骨转录因子Osterix(OSX)表达的增加是必不可少的。我们的建议将解决一个尚未回答的关键问题，即抗氧化剂AA如何调节核因子的转录活性。为此，我们有了新的令人兴奋的初步数据，证明了Pro-2参与介导了OBS中的AA效应。根据已发表的P53抑制OB分化、OSX表达和骨发育的数据，以及我们的初步数据表明AA通过PHD2依赖机制降低P53蛋白水平，我们将通过检验3个假设来检验AA调节OSX表达的模型，该模型涉及PHD2和p53。为了验证假设1，PHD2通过以不依赖于HIF-11的方式调节OSX的表达来介导AA对OB分化和骨形成(BF)的影响，我们将：1)证实OB产生的PHD2通过腺病毒(Ad)-Cre介导的PHD2的破坏参与介导AA对OB分化的影响；2)建立AA诱导的OB分化过程中OSX的表达是由PHD2介导的，而不是HIF11；c)利用CRE/loxP技术确定PHD2在条件中断PHD2调节BF中的作用；4)通过建立AA缺乏的PHD2条件性KO小鼠，证实AA修复SFX小鼠BF缺乏症需要PHD2。为了验证假设2，AA激活PHD2会导致P53的Pro羟化，从而调节蛋白质小体的降解，我们将：1)确定AA通过PHD2调节P53的Pro羟化；2)确定PHD2介导的PHD2介导的P53的Pro羟化是否受到泛素介导的蛋白体降解；以及3)通过突变P53中的潜在羟化位点来确定Pro羟化的位置，并确定突变的P53是否抵抗蛋白小体的降解，以及是否比WT更有效地抑制OSX的表达。为了检验假设3，P53与OSX启动子的抗氧化反应元件(ARE)相互作用，并与bZIP转录因子竞争抑制OSX的超越，我们将：1)用来自PHD2条件KO和WT小鼠的OBS-RACE进行EMSA和CHIP检测，证实P53与OSX基因启动子的Are结合，并且竞争已知与ARE相互作用的bZIP转录因子家族的成员；2)在OSX近端的启动子内突变可能的ARE，并评价P53的S对OBS启动子活性的影响。我们证实了PHD2通过P53依赖和HIF11不依赖的机制介导AA对OSX表达和OB分化的影响的假设，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.