Prolyl Hydroxylases, Epigenetics and Osteoarthritis

 描述(由申请人提供)： 项目概述/摘要该项目的长期目标集中在确定维生素C在骨骼生物学中的作用和作用机制，因为维生素C缺乏会导致骨骼骨折。在上一次资助期间，我们发现了Pro羟基酶结构域蛋白(PhDS)除了介导维生素C对成骨细胞(OB)的影响外，还在调节关节软骨(AC)发育方面发挥了新的作用。通过在OBS和软骨细胞中进行PHD2基因条件敲除(CKO)的小鼠，我们发现OBS中PHD2基因的缺失导致骨量减少，而PHD2的软骨细胞特异性KO导致软骨内骨形成(EBF)增加而导致骨小梁数量急剧增加，从而暗示PHD2在这两种细胞类型中的不同作用和机制。新的初步数据显示，PHD2和PHD3分别在AC的浅层(SZ)和中层(MZ)有不同的表达，在SZ的AC软骨细胞前体细胞向MZ的分化软骨细胞的转变具有EBF能力，PHD2和PHD3的相对表达水平密切相关。此外，我们的数据表明，PHD2和PHD3通过精确控制低氧诱导因子1α和HIF2α水平以及涉及5-甲基胞嘧啶(5-MC)羟化的表观遗传机制来调控AC软骨细胞。基于这些数据，我们在这项竞争性更新申请中的主要目标是阐明PHD2和PHD3在AC发展和骨关节炎(OA)发病机制中的作用，因为骨关节炎在美国是一个重大的公共卫生问题，在没有可用的治疗方法的情况下造成巨大的经济负担。我们将使用新的遗传小鼠模型来验证AC中PHD2/PHD3表达异常通过涉及HIF信号和基因组活性的表观遗传修饰而参与OA发病机制的预测。在特定的目标1中，我们将检验一种假设，即在AC前体细胞中PHD2的表达缺失通过促进HIF1α信号来损害AC的发育和维持。我们将在表达Prg4的AC祖细胞中产生PHD2表达中断的小鼠，并检测AC表型随年龄的变化以及对内侧半月板损伤的反应，并确定PHD2CKO小鼠的AC表型是否通过中断AC祖细胞中HIF1α的表达而消失。在具体目标2中，我们将测试 假设在分化的软骨细胞中PHD3的表达缺失通过促进HIF2α信号转导来调节AC表型。我们将在表达分化软骨细胞的聚集素中产生PHD3表达中断的小鼠，并确定PHD3CKO小鼠的AC表型是否因HIF2α信号中断而被取消。在特定的目标3中，我们将检验PHD2和PHD3通过调节5-MC的羟化来部分通过表观遗传控制基因组活性来调节AC前体细胞向分化软骨细胞的转变的假设。我们建议的研究的成功完成将为PHD2和PHD3调节AC的途径提供重要的信息，并可能为治疗老年退伍军人中流行的骨性关节炎提供新的基于PHD的药物靶点。

 DESCRIPTION (provided by applicant): Project Summary/Abstract The long-term goals of this Merit project are focused on characterizing the roles and mechanisms of actions of vitamin C in skeletal biology since its deficiency induces skeletal fractures. During the last funding period, we identified novel roles fo prolyl hydroxylase domain proteins (PHDs) in regulating articular cartilage (AC) development besides mediating vitamin C effects on osteoblasts (OBs). By using mice with a conditional knockout (cKO) of the Phd2 gene in OBs and chondrocytes, we determined that loss of Phd2 in OBs resulted in osteopenia while chondrocyte-specific KO of Phd2 lead to a dramatic increase in trabecular bone mass caused by increased endochondral bone formation (EBF), thus suggesting distinct roles and mechanisms for PHD2 in the two cell types. New preliminary data described in this proposal show that Phd2 and Phd3 are differentially expressed in the superficial zone (SZ) and middle zone (MZ) of AC, respectively, and that transition of AC chondrocyte progenitors in the SZ into differentiating chondrocytes in the MZ capable of EBF is tightly regulated by relative levels of Phd2 and Phd3 expression. Furthermore, our data show that PHD2 and PHD3 regulate AC chondrocytes by precise control of hypoxia-inducible factor (HIF)1α and HIF2α levels and by an epigenetic mechanism involving hydroxylation of 5- methyl cytosine (5-mC). Based on these data, our major goal in this competitive renewal application is to elucidate the role of Phd2 and Phd3 in AC development and in the pathogenesis of osteoarthritis (OA), since OA is a significant public health problem in the U.S., posing a substantial financial burden with no available therapies for treatment. We will use new genetic mouse models to test the prediction that dysregulation of Phd2/Phd3 expression in AC contributes to the pathogenesis of OA by involving HIF signaling and epigenetic modification of genome activity. In Specific Aim 1, we will test the hypothesis that loss of Phd2 expression in AC progenitors impairs development and maintenance of AC by promoting HIF1α signaling. We will generate mice in which expression of Phd2 is disrupted in Prg4 expressing AC progenitors and examine the AC phenotype as a function of age and in response to a destabilization of medial meniscus (DMM) injury and determine if the AC phenotype in Phd2 cKO mice is abolished by disruption of Hif1α expression in AC progenitors. In Specific Aim 2, we will test the hypothesis that loss of Phd3 expression in differentiating chondrocytes regulates the AC phenotype by promoting HIF2α signaling. We will generate mice in which expression of Phd3 is disrupted in aggrecan expressing differentiating chondrocytes and determine if the AC phenotype in Phd3 cKO mice is abolished by disruption of HIF2α signaling. In Specific Aim 3, we will test the hypothesis that Phd2 and Phd3 regulate transition of AC progenitors into differentiating chondrocytes in part via epigenetic control of genome activity via regulating hydroxylation of 5-mC. Successful completion of our proposed studies will provide important information on the pathway by which Phd2 and Phd3 regulate AC and could provide novel PHD-based drug targets for treatment of OA prevalent in aging veterans.