Prolyl Hydroxylases, Epigenetics and Osteoarthritis

 描述（由申请人提供）： 项目摘要/摘要本Merit项目的长期目标是重点描述维生素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。在上一个资助期间，我们确定了脯氨酰羟化酶结构域蛋白（PHDs）在调节关节软骨（AC）发育中的新作用，除了介导维生素C对成骨细胞（OB）的作用。通过使用OB和软骨细胞中Phd 2基因的条件性敲除（cKO）小鼠，我们确定OB中Phd 2的缺失导致骨质减少，而软骨细胞特异性敲除Phd 2导致由增加的软骨内骨形成（EBF）引起的小梁骨量急剧增加，从而表明PHD 2在两种细胞类型中的不同作用和机制。本提案中描述的新的初步数据表明，Phd 2和Phd 3分别在AC的浅表区（SZ）和中间区（MZ）中差异表达，并且SZ中的AC软骨细胞祖细胞向能够进行EBF的MZ中的分化软骨细胞的转变受到Phd 2和Phd 3表达的相对水平的严格调控。此外，我们的数据表明，PHD 2和PHD 3通过精确控制缺氧诱导因子（HIF）1α和HIF 2 α水平以及涉及5-甲基胞嘧啶（5-mC）羟基化的表观遗传机制来调节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Phd 2和Phd 3在AC发展和骨关节炎（OA）发病机制中的作用，因为OA是美国的一个重要公共卫生问题，造成了巨大的经济负担，没有可用的治疗方法。我们将使用新的遗传小鼠模型来测试AC中Phd 2/Phd 3表达的失调通过涉及HIF信号传导和基因组活性的表观遗传修饰而有助于OA发病机制的预测。在具体目标1中，我们将检验AC祖细胞中Phd 2表达缺失通过促进HIF 1 α信号传导损害AC发育和维持的假设。我们将产生在表达Prg 4的AC祖细胞中Phd 2表达被破坏的小鼠，并检查AC表型作为年龄的函数和对内侧半月板（DMM）损伤不稳定的响应，并确定Phd 2 cKO小鼠中的AC表型是否通过破坏AC祖细胞中的Hif 1 α表达而消除。在具体目标2中，我们将测试 假设在分化的软骨细胞中Phd 3表达的缺失通过促进HIF 2 α信号传导调节AC表型。我们将产生在表达聚集蛋白聚糖的分化软骨细胞中Phd 3表达被破坏的小鼠，并确定Phd 3 cKO小鼠中的AC表型是否通过破坏HIF 2 α信号传导而消除。在具体目标3中，我们将检验Phd 2和Phd 3通过调节5-mC的羟基化，通过基因组活性的表观遗传控制，部分地调节AC祖细胞向分化的软骨细胞的转变的假设。我们提出的研究的成功完成将提供关于Phd 2和Phd 3调节AC的途径的重要信息，并可以为治疗老年退伍军人中流行的OA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.