Role of Ephrins in Osteoblast Differentiation

描述（由申请人提供）：骨骼活力需要在一生中精确调节骨合成，而伴随其失败的灾难性病理状况提出了重大的公共卫生挑战。然而，来自周围组织的信号调节骨合成的机制尚不清楚，并且这种知识的缺乏阻碍了治疗骨生长缺陷疾病的能力。因此，该小组的长期目标是了解控制成骨细胞（OB）（骨形成细胞）分化的分子机制，以便更好地操纵骨合成来治疗骨疾病。最近的文献工作和本文提供的初步数据表明并支持受体酪氨酸激酶 Eph 家族成员及其配体肝配蛋白在接触介导的 OB 分化促进中发挥核心作用。具体来说：1) Ephs 和肝配蛋白在体内成骨的离散层中表达，2) 用可溶性重组肝配蛋白处理 OB 会增加 OB 特异性基因表达和骨合成。基于这一证据，该提案提出了一个中心假设：Eph 信号通过激活特定的成骨细胞基因启动子元件来调节骨生长。拟议研究的基本原理是，更深入地了解这些受体如何参与接触介导的 OB 分化，将揭示未来治疗中利用的控制点。这一假设将通过完成两个具体目标来解决：1) 确定激活或阻断 Eph 信号传导对正常和有缺陷的骨形成的影响，2) 确定特定启动子元件对 OB 基因激活的贡献。第一个目标的研究将把重组肝配蛋白应用于成熟的颅骨骨生长模型，以刺激成骨。相反，Eph胞外结构域蛋白的应用将用于竞争性抑制内源性肝配蛋白，从而阻断成骨。这些研究具有基础科学和转化相关性，因为它们将确定 Eph/ephrin 信号传导在骨缺陷治疗中的重要性和潜在应用。第二个目标下的研究将使用先前表征的 OB 特异性启动子驱动的荧光素酶报告基因来识别这些启动子内的顺式作用元件，这些元件负责肝配蛋白诱导 OB 中的基因转录。这将导致鉴定出可能适合在临床环境中操作的转录因子。这项工作具有创新性，因为它将揭示 Ephs 在基因调控中的全新作用，并将阐明成骨调控的新机制。它将产生重大影响，因为它有可能为骨疾病的治疗提供新的治疗靶点。 公共健康相关性：计划中的研究提出了 Eph/ephrin 信号在基因表达调节中的迄今为止未被描述的作用。它们对公共卫生有特殊的应用，因为它们解决了在致残性骨疾病的发病机制中失调的成骨细胞分化的基本机制。因此，它们有望为这些疾病的临床治疗的进步做出贡献。此外，由于肝配蛋白几乎参与器官发生的每个阶段，因此这项工作有可能对发育和分子生物学领域产生更广泛的影响。

DESCRIPTION (provided by applicant): Skeletal viability requires precise regulation of bone synthesis throughout life, and the catastrophic pathological conditions that accompany its failure present a major public health challenge. Nevertheless, the mechanisms through which synthesis of bone is regulated by signals from the surrounding tissues are not understood, and this lack of knowledge hampers the ability to treat diseases of defective bone growth. The long-term goal of this group is therefore to understand the molecular mechanisms that control differentiation of the osteoblast (OB), the bone-forming cell, so as to better manipulate bone synthesis in the treatment of bone diseases. Recent work in the literature and preliminary data presented herein suggest and support a central role for the contact-mediated promotion of OB differentiation by members of the Eph family of receptor tyrosine kinases and their ligands, the ephrins. Specifically:1) Ephs and ephrins are expressed in discrete layers of osteogenesis in vivo, and 2) treatment of OBs with soluble recombinant ephrin increases OB-specific gene expression and bone synthesis. Based on this evidence, this proposal advances the central hypothesis that Eph signaling regulates bone growth through the activation of specific osteoblast gene promoter elements. The rationale for the proposed studies is that a deeper understanding of how these receptors participate in contact-mediated OB differentiation will reveal control points for exploitation in future therapies. This hypothesis will be addressed through completion of two specific aims:1) Determine the effect of activating or blocking Eph signaling on normal and defective bone formation, and 2) Define the contribution of specific promoter elements to OB gene activation. Studies under the first aim will apply recombinant ephrins to a well-established calvarial model of bone growth to stimulate osteogenesis. Conversely, application of Eph extracellular domain protein will be used to competitively inhibit endogenous ephrins and thus block osteogenesis. These studies have both basic science and translational relevance in that they will establish the importance of, and potential application for, manipulation of Eph/ephrin signaling in the treatment of bone defects. Studies under the second aim will use previously characterized OB-specific promoter-driven luciferase reporters to identify the cis-acting elements within these promoters that are responsible for ephrin induction of gene transcription in OBs. This will lead to identification of transcription factors that may be amenable to manipulation in a clinical setting. The proposed work is innovative because it will uncover an entirely new role for Ephs in gene regulation, and will illuminate a novel mechanism for the regulation of osteogenesis. It will make a significant impact because of its potential to offer new therapeutic targets for the treatment of bone diseases. Public Health Relevance: The planned studies propose a heretofore-undescribed role for Eph/ephrin signaling in the regulation of gene expression. They have particular application to the public health in that they address basic mechanisms of osteoblast differentiation that are dysregulated in the pathogenesis of crippling bone diseases. Thus, they can be expected to contribute to advances in clinical treatment of these afflictions. Further, as ephrins are involved in virtually every stage of organogenesis, this work has the potential for broader impact on the fields of developmental and molecular biology.