Integrative genomics to define osteocyte differentiation, regulation and function

描述（由申请人提供）：骨细胞是成骨细胞来源的细胞，逐渐埋藏在矿化基质中，其功能是调节骨骼稳态；它们的重要性体现在它们相对丰富的数量和异常长的寿命上。令人惊讶的是，尽管已知骨细胞相对于成骨细胞前体表现出独特的基因表达模式，因此，表现出独特的形式和功能，但对协调它们从成骨细胞转变的分子机制或对此负责的表观基因组和规则决定因素知之甚少。此外，尽管已知Wnt信号是成骨细胞和骨细胞活性的主要差异调节因子，但构成该途径细胞特异性活性的大多数基因靶点以及TCF/LEF/b-catenin在两种细胞类型中调节这些靶点的机制仍不清楚。因此，前两个目标的目标如下。目的1：确定潜在的表观基因组和规律机制以及相关的分子决定因素，这些决定因素在全基因组范围内负责体外和体外成骨细胞向骨细胞的转化。目的2：检查和对比Wnt信号通路对成骨细胞和成熟骨细胞特异性基因表达模式的影响，并评估这些基因亚群在体外和离体中由b-连环蛋白直接调节并受PTH和125 (OH)2D3影响的分子机制。我们和其他人的初步数据表明，在骨细胞转化过程中，许多基因上调，包括Sost、Fgf23、Tnfsf11和Enpp1/3。Sost基因编码Wnt通路拮抗剂硬化蛋白（sclerostin， SCL），然而，由于其对骨形成的核心生物学作用、对疾病的影响以及作为潜在治疗靶点的相关性，Sost基因至关重要。在了解Sost调控方面取得了进展，部分原因是发现下游Sost增强子的缺失改变了Sost的表达，并导致了Van Buchem病。然而，我们的初步数据表明，复杂性大大增加，因此支持本建议的最终目标。目的3：在体外和体内模型中，评估Sost在表观基因组和规律水平上的基础和调节骨细胞表达的分子机制。骨细胞在骨骼中发挥着独特的作用，并以内分泌方式发挥作用，既可以调节局部旁分泌因子（如硬化素），也可以调节系统活性激素（如FGF23）。骨细胞功能异常引起的病理后果可能是灾难性的。从拟议的研究中产生的详细的基本见解可能为多种疾病的治疗干预提供新的途径

DESCRIPTION (provided by applicant): Osteocytes are osteoblast-derived cells that are progressively entombed in mineralized matrix where they function to regulate skeletal homeostasis; their importance is highlighted both by their relative abundance and by their exceptionally long lifespan. Surprisingly, although osteocytes are known to manifest unique gene expression patterns relative to their osteoblast precursors and, as a consequence, display unique form and function, little is known of the molecular mechanisms that orchestrate their transition from the osteoblast or of the epigenomic and regulomic determinants that are responsible. In addition, although Wnt signaling is known to be a major but differential regulator of osteoblast and osteocyte activities, the majority of the gene targets that comprise the cell-specific activities of this pathway and the mechanisms through which TCF/LEF/b-catenin function to modulate these targets in both cell types remain unknown. Accordingly, the objectives of the first two aims are as follows. Aim 1: Identify the underlying epigenomic and regulomic mechanisms and associated molecular determinants that are responsible on a genome-wide scale for the osteoblast to osteocyte transition both in vitro and ex vivo. Aim 2: Examine and contrast the impact of the Wnt signaling pathway on osteoblast- and mature osteocyte-specific gene expression patterns and assess the molecular mechanisms through which these gene subsets are modulated directly by b-catenin and impacted by PTH and 1,25(OH)2D3 both in vitro and ex vivo. Our preliminary data and that of others have shown that a number of genes are upregulated during the osteocyte transition, including Sost, Fgf23, Tnfsf11, and Enpp1/3. The Sost gene, which encodes the Wnt pathway antagonist sclerostin (SCL), is of primary importance, however, due to its central biological actions on bone formation, its impact in disease, and its relevance as a potential therapeutic target. Progress has been made in understanding Sost regulation, prompted in part by the discovery that deletion of a downstream Sost enhancer alters Sost expression and is responsible for Van Buchem disease. Our preliminary data, however, suggest significant additional complexity, thus supporting the final objective of this proposal. Aim 3: Assess the molecular mechanisms that underlie both basal and regulated osteocyte expression of Sost at both epigenomic and regulomic levels in both in vitro and in vivo models. Osteocytes play unique roles in the skeleton and act in endocrine fashion to elaborate both local paracrine factors such as sclerostin and systemically active hormones such as FGF23. Pathologic consequences arising from aberrant osteocyte function can be catastrophic. Detailed basic insights arising from the proposed studies are likely to provide new routes of therapeutic intervention for a multiplicity of diseases that affect the skeleton.