The Role of Mef2C in Bone

项目摘要 全世界每年有超过2亿人患有骨质疏松症，尤其是绝经后妇女， 大约有900万处骨折，或者说每3秒钟就有一处骨折。这是一种骨密度和强度降低的疾病， 减少到骨骼不能充分发挥其支持功能的程度，增加了 骨折，并导致老年人的发病率和死亡率。此外，其他骨骼变薄 诸如骨质减少、成骨不全和肾性骨营养不良的疾病是普遍的， 迫切需要促进骨骼健康的有效疗法。沿着WNT信号通路的突变 已被证明在骨代谢中起关键作用，引发了人们对确定和 可能利用WNT信号传导在骨中的作用作为治疗骨质疏松症的新的治疗方法。 骨质疏松症、其他LBM疾病和骨脆性增加。我们的研究项目已经产生了 Mef 2C和Sost作为WNT信号通路的关键组分相互联系的重要数据， 成骨细胞/骨细胞。对Mef 2C、ECR 5和Sost的遗传分析已经确定了这些蛋白质和Sost’s 调节元件作为骨稳态的关键介质。在这个建议中，我们打算精确地指出 什么样的分子功能与骨中的哪种细胞类型相关，以解剖出细胞类型特异性 Mef 2C和Sost在骨代谢过程中对不同功能的贡献。特别重点将放在 目的1：研究Mef 2C在破骨细胞中的作用;使用不同Cre重组酶转基因小鼠的组合，我们将 删除破骨细胞和成骨细胞中的Mef 2C，以确定：a）Mef 2C是否通过激活 促进破骨细胞中骨吸收的基因和抑制成骨细胞中骨形成的基因; B）如果 Mef 2C KO通过独立于Sost的机制引起高骨量。在目标2中，我们将确定 在破骨细胞中，Mef 2C直接或间接控制能量代谢基因的转录。通过 我们将确定RNAseq、ChIPseq、增强子验证、siRNA和Mef 2C过表达的组合 （1）Ppargc 1 β和ppargc 1 α是否是破骨细胞中Mef 2C的直接转录靶点;（2）Mef 2C 与Ppargc 1 β和/或ppargc 1 α发生物理相互作用，与破骨细胞中类似的DNA元件结合，以及是否 (3)162个能量代谢基因在Mef 2CcKO中下调; Ctsk-Cre小鼠是直接转录靶点 Mef 2C的在目标3中，我们将鉴定推定的Mef 2C破骨细胞和成骨细胞增强剂，并在 体外，在细胞系模型中。将使用经验证的增强子及其转录靶基因 来构建在骨代谢过程中被调节的转录网络。我们的首要目标是 了解Mef 2C如何通过调节破骨细胞和成骨细胞基因来促进骨代谢 表达，并确定参与这一过程的分子;这项工作可能最终导致 发现新的候选分子，可以治疗靶向改善人类骨骼健康。

Project Summary Worldwide osteoporosis affects over 200 million people annually, particularly postmenopausal women who suffer ~9 million fractures or one fracture every 3 seconds. It is a disease in which bone density and strength is diminished to a point where the skeleton cannot adequately perform its support functions, increasing the risk of fractures and contributing to substantial morbidity and mortality in the elderly. In addition, other bone thinning disorders such as osteopenia, osteogenesis imperfect and renal osteodystrophy are prevalent, creating an urgent need for effective therapies that promote bone health. Mutations along the WNT signaling pathways have been shown to play key roles in bone metabolism, triggering tremendous interest in determining and possibly exploiting the role of WNT signaling in bone as a new therapeutic approach for the treatment of osteoporosis, other diseases with LBM and increased bone fragility. Our research program has generated important data interconnecting Mef2C and Sost as critical components of WNT signaling pathway in osteoblasts/osteocytes. Genetic analysis of Mef2C, ECR5 and Sost has established these proteins and Sost's regulatory element as key mediators of bone homeostasis. In this proposal we intended to pinpoint precisely what molecular functions are associated with which cell type(s) in bone to dissect out cell-type specific contributions of Mef2C and Sost to distinct functions during bone metabolism. Particular focus will be given in Aim 1 to Mef2C role in osteoclasts; using a combination of different Cre-recombinase transgenic mice we will delete Mef2C in osteoclasts and osteoblasts to determine: a) if Mef2C has dual roles in bone by activating genes that promote bone resorption in osteoclasts and genes that inhibit bone formation in osteoblasts; b) if Mef2C KO causes high bone mass by mechanism independent of Sost. In Aim 2 we will determine whether Mef2C directly or indirectly controls the transcription of energy metabolism genes, in osteoclasts. Through a combination of RNAseq, ChIPseq, enhancer validation, siRNA and overexpression of Mef2C we will determine whether (1) Ppargc1β and ppargc1α are direct transcriptional targets of Mef2C in osteoclasts; (2) Mef2C physically interact with Ppargc1β and/or ppargc1α to bind to similar DNA elements in osteoclasts, and whether (3) 162 energy metabolism genes down-regulated in Mef2CcKO; Ctsk-Cre mice are direct transcriptional targets of Mef2C. In Aim 3 we will identify putative Mef2C osteoclast and osteoblast enhancers, and validate them in vitro, in cell line models. Validated enhancers in combination with their transcriptional target genes will be used to build transcriptional networks that are modulated during bone metabolism. Our overarching goal is to understand how Mef2C contributes to bone metabolism by regulating osteoclast and osteoblast gene expression and to identify the molecules involved in this process; this work could ultimately lead to the discovery of new candidate molecules that could be therapeutically targeted to improve human bone health.