Role of Ephrins in Osteoblast Differentiation

描述(由申请人提供)：骨骼的生存能力需要在一生中对骨合成进行精确的调节，而伴随其失败而来的灾难性病理条件是一个重大的公共卫生挑战。然而，骨合成受周围组织信号调控的机制尚不清楚，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骨形成细胞成骨细胞(OB)分化的分子机制，以便在骨疾病的治疗中更好地操纵骨合成。最近的文献和初步数据表明，并支持Eph受体酪氨酸激酶家族成员及其配体ephins通过接触方式促进OB分化的核心作用。具体地说：1)Ephs和Eparin在体内成骨的不同层次中表达，2)用可溶性重组Ephin处理OB可以增加OB特异性基因的表达和骨合成。基于这一证据，这一建议提出了中心假设，即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，深入了解这些受体如何参与接触介导的OB分化，将揭示未来治疗中的控制点。这一假说将通过完成两个具体目标来解决：1)确定激活或阻断Eph信号对正常和缺陷骨形成的影响；2)确定特定启动子元件对OB基因激活的贡献。在第一个目标下的研究将把重组肾上腺素应用于已建立的颅骨生长模型，以刺激成骨。相反，应用Eph胞外区蛋白将被用来竞争性地抑制内源性肾上腺素，从而阻止成骨。这些研究既具有基础科学意义，又具有翻译相关性，因为它们将确立Eph/ePhin信号在骨缺损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描述的OB特异性启动子驱动的荧光素酶报告，以确定这些启动子中负责ePhin诱导OB基因转录的顺式作用元件。这将导致识别可能在临床环境中易于操作的转录因子。这项拟议的工作具有创新性，因为它将揭示Ephs在基因调控中的全新角色，并将阐明一种新的成骨调控机制。它将产生重大影响，因为它有可能为骨骼疾病的治疗提供新的治疗靶点。 与公共卫生的相关性：计划中的研究提出了一个迄今为止未被描述的Eph/EPhin信号在基因表达调控中的作用。它们在公共卫生方面有特殊的应用，因为它们解决了成骨细胞分化的基本机制，这些机制在致残性骨骼疾病的发病机制中受到失调。因此，可以预期它们将为这些疾病的临床治疗做出贡献。此外，由于eparin几乎参与器官发生的每个阶段，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.