Molecular Control of Bone Formation

摘要 我们计划的研究将调查表观遗传酶调节谱系的中心假设- 骨形成细胞在体内的定型和成熟。我们的研究将为骨骼的新策略提供信息， 旨在降低骨折风险的合成代谢疗法。目前的治疗具有主要的临床副作用， 患者限制。然而，随着美国人口的老龄化， 开发新的策略，促进骨积累。此外，还有一系列的短期合成代谢 应用（例如，脊柱融合、放射性骨丢失），将受益于基于新机制的 接近。拟议的工作将提供（i）利用表观遗传药物促进 在翻译前环境中的骨累积，而且（ii）对染色质作用的基本见解 异染色质化控制骨形成。 目的1将评估有条件切除组蛋白基因的体内骨刺激作用 甲基转移酶EZH 2在出生后发育过程中和在睾丸成熟小鼠中的表达，其中EZH 2 基因在间充质干细胞或成骨细胞中缺失。这些研究将揭示基因丢失是否 EZH 2刺激骨积累，是骨保护性的（在卵巢切除小鼠中雌激素耗尽后），和/或 加速骨骼愈合这些遗传学研究将根据我们的初步数据进行后续研究， 具有确定的安全性特征的药理学抑制剂（例如，EZH 2抑制剂GSK 126）可用于 促进体内骨生成和体外成骨细胞分化。 目的2将检查EZH 2失活的骨刺激作用的分子基础。使用 基因组学方法（ChIP-seq和RNA-seq），我们将定义和功能测试信号分子， 对EZH 2抑制有反应的基因调节因子。我们还将专门调查有吸引力的 EZH 2抑制通过增强内源性旁分泌启动持续骨合成代谢反应的模型 成骨配体的信号传导（例如，BMP、WNT和PTH）。我们将特别着重研究 我们新观察到GSK 126和BMP 2在成骨细胞成熟中协同作用的分子基础。 我们的研究结果具有重要的临床影响，通过产生遗传支持考虑表观遗传 促进新骨形成的药物。这些研究的概念创新将通过分子生物学的方法来实现。 分析EZH 2的表观遗传调节作用，其允许定义早期机制事件， 控制成骨分化和成骨细胞成熟。

ABSTRACT Our proposed studies will investigate the central hypothesis that epigenetic enzymes regulate lineage- commitment and maturation of bone forming cells in vivo. Our studies will inform new strategies for bone anabolic therapies aimed at reducing fracture risk. Current therapies have major clinical side-effects and patient restrictions. Yet, with the aging demographics of the US population, there is a significant urgency to develop new strategies that promote bone accrual. In addition, there are a range of short term anabolic applications (e.g., spine fusion, radiation induced bone loss) that would benefit from new mechanism-based approaches. The proposed work will provide (i) proof of concept for leveraging epigenetic drugs to promote bone accrual in a pre-translational setting, but also (ii) fundamental insights into the role of chromatin heterochromatinization in controlling bone formation. Aim 1 will assess the in vivo bone stimulatory effects of conditional ablation of the gene for histone methyl transferase EZH2 during post-natal development and in skeletally mature mice in which the EZH2 gene is deleted in mesenchymal stem cells or osteoblasts. These studies will reveal whether genetic loss of EZH2 stimulates bone accrual, is osteoprotective (after estrogen-depletion in ovariectomized mice), and/or accelerates bone healing. These genetic studies will follow-up on our preliminary data indicating that pharmacological inhibitors with established safety profiles (e.g., EZH2 inhibitor GSK126) can be used to promote bone accrual in vivo and osteoblast differentiation in vitro. Aim 2 will examine the molecular basis for the bone stimulatory effects of EZH2 inactivation. Using genomics approaches (ChIP-seq and RNA-seq), we will define and functionally test signaling molecules and gene regulatory factors that respond to EZH2 inhibition. We will also specifically investigate the attractive model that EZH2 inhibition initiates a sustained bone anabolic response by enhancing endogenous paracrine signaling of osteogenic ligands (e.g., BMP, WNT and PTH). We will focus specifically on examining the molecular basis for our new observation that GSK126 and BMP2 synergize in osteoblast maturation. Our findings have important clinical ramifications by generating genetic support for considering epigenetic drugs in promoting new bone formation. Conceptual innovation of these studies will be obtained by molecular analysis of the epigenetic regulatory role for EZH2 which permits definition of early mechanistic events that control osteogenic differentiation and osteoblast maturation.