Demystify Heparan Sulfate–Sclerostin Interactions in Bone Formation

项目摘要 硬化素是一种有效的骨形成抑制剂，已被证明是一种有价值的药物。 治疗骨质疏松症的靶点。从机制上讲，硬化素通过与LRP5/6结合发挥作用 成骨细胞系细胞拮抗规范的Wnt信号，从而负调控 骨形成。据推测，在骨细胞分泌硬化素后，它们就会到达靶点。 成骨细胞在骨表面扩散。然而，到目前为止，人们仍然不知道如何 分泌的硬化素在细胞表面和细胞外基质中受到调节。致信地址 针对硬化素生物学上的这一重大空白，我们重点研究了硬化素-硫酸肝素(HS)。 互动。HS是一种普遍存在于细胞表面和细胞外的糖胺多糖 基质，已知结合硬化素，并可能调节生物活性和扩散的硬化素。 我们的中心假设是HS可以调节硬化素在骨中的生物学功能 队形。为了检验这一假设，我们的总体目标是阐明HS是如何相互作用的 与硬化素的相互作用以及HS-硬化素相互作用如何调节骨形成。我们计划追查 以下两个具体目标：目的1.确定HS-skerostin的生物学意义 体外相互作用。我们推测HS有助于将硬化素集中在成骨细胞上 并通过形成三元组分促进硬化素与其受体LRP5/6的结合 很复杂。我们还假设HS在细胞表面提供了一个硬化素的储存库 在它被分泌后，它可以保护骨细胞，并保护它免受蛋白质降解的影响。我们会操纵 HS-skerostin在成骨细胞和骨细胞表面的生化相互作用 确定HS调节硬化素在这些细胞中的功能的机制 上下文。目的2.确定HS-skerostin相互作用在体内骨形成中的作用。我们的 工作假说是，抑制HS-skerostin相互作用会削弱抑制效力 向LRP5/6转化为硬化素，从而促进骨形成。使用一种新型的硬化剂 在敲击小鼠品系中，我们将检查改变HS-skerostin相互作用的后果 在体内的骨形成中。我们的贡献将是重大的，因为我们将确定多个 HS调节硬化素的分子机制并阐明这种相互作用 调节骨形成。拟议的实验结果将极大地推动我们的 了解硬化素对成骨细胞和骨细胞的细胞调节作用 阐明了HS在系统中的作用。重要的是，这些结果预计会有积极的影响。 翻译的影响，因为通过确定HS如何调节硬化剂的生物利用度，我们 可能为硬化素在骨科疾病中的应用提供新的视角。

Project Summary Sclerostin is a potent inhibitor of bone formation and has been shown to be a valuable drug target for treating osteoporosis. Mechanistically, sclerostin functions by binding to LRP5/6 on the osteoblast lineage cells to antagonize canonical Wnt signaling, thus negatively regulates bone formation. Presumably, after sclerostin is secreted by osteocytes, they reach the target osteoblasts at the bone surfaces by diffusion. However, to date it remains unknown how secreted sclerostin is regulated on the cell surface and in the extracellular matrix. To address this significant gap in sclerostin biology, we focus our study on sclerostin–heparan sulfate (HS) interaction. HS, a universal glycosaminoglycan found at the cell surface and in the extracellular matrix, is known to bind sclerostin and might regulate the bioactivity and diffusion of sclerostin. Our central hypothesis is that HS can regulate the biological functions of sclerostin in bone formation. To test this hypothesis, our overall objective here is to elucidate how HS interacts with sclerostin and how HS–sclerostin interaction regulates bone formation. We plan to pursue the following two specific aims: Aim 1. Determine the biological significance of HS–sclerostin interactions in vitro. We postulate that HS helps concentrate sclerostin on the osteoblast cell surface and facilitates the binding of sclerostin to its receptor LRP5/6 by forming ternary complex. We also hypothesize that HS serves a storage depot of sclerostin on the cell surface of osteocytes after it is secreted, and protects it from proteolytic degradation. We will manipulate HS–sclerostin interactions biochemically at the surface of both osteoblasts and osteocytes to determine the mechanisms by which HS regulates the function of sclerostin in these cellular contexts. Aim 2. Determine the role of HS–sclerostin interaction in bone formation in vivo. Our working hypothesis is that dampening HS–sclerostin interactions impairs the inhibitory potency of sclerostin towards LRP5/6, which leads to enhanced bone formation. Using a novel sclerostin knock-in mouse strain, we will examine the consequence of altering HS–sclerostin interactions in bone formation in vivo. Our contribution will be significant because we will identify multiple molecular mechanisms by which HS regulates sclerostin and elucidate how such interactions regulate bone formation. Results from the proposed experiments will substantially advance our understanding of the cellular regulation of sclerostin on both osteoblasts and osteocytes by elucidating the role of HS in the system. Importantly, these results are expected to have positive translational impact because by identifying how HS regulates the bioavailability of sclerostin, we may be able to provide new perspective for manipulating sclerostin in bone diseases.