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，硬化蛋白通过结合LRP 5/6来发挥作用， 成骨细胞谱系细胞拮抗经典Wnt信号传导，从而负调节 骨形成据推测，骨细胞分泌sclerostin后， 成骨细胞在骨表面扩散。然而，迄今为止， 分泌的硬化蛋白在细胞表面和细胞外基质中受到调节。解决 由于sclerostin生物学的这一重大空白，我们将研究重点放在sclerostin-硫酸乙酰肝素（HS）上。 互动HS，一种在细胞表面和细胞外基质中发现的通用糖胺聚糖， 已知基质结合硬骨素并可能调节硬骨素的生物活性和扩散。 我们的中心假设是，HS可以调节骨硬化素的生物学功能 阵为了验证这一假设，我们的总体目标是阐明HS如何相互作用 以及HS-sclerostin相互作用如何调节骨形成。我们计划继续 以下两个具体目标：目标1。确定HS-硬化蛋白的生物学意义 体外相互作用我们假设HS有助于将硬化蛋白集中在成骨细胞上， 表面，并通过形成三元结构促进硬化蛋白与其受体LRP 5/6的结合。 复杂.我们还假设HS在细胞表面充当硬化蛋白的储存库 骨细胞分泌后，并保护它免受蛋白水解降解。我们将操纵 HS-硬化蛋白在成骨细胞和骨细胞表面的生物化学相互作用， 确定HS调节这些细胞中硬化素功能的机制 contexts.目标二。确定HS-硬化蛋白相互作用在体内骨形成中的作用。我们 工作假设是抑制HS-硬化蛋白相互作用会损害抑制效力 硬化蛋白的LRP 5/6，这导致骨形成增强。使用一种新的硬化素 敲入小鼠品系，我们将研究改变HS-硬化蛋白相互作用的后果 在体内骨形成中的作用。我们的贡献将是重大的，因为我们将确定多个 HS调节sclerostin的分子机制，并阐明这种相互作用 调节骨形成。从拟议的实验结果将大大推进我们的 理解sclerostin对成骨细胞和骨细胞的细胞调节， 阐明HS在系统中的作用。重要的是，这些结果预计将产生积极的影响。 翻译的影响，因为通过确定HS如何调节sclerostin的生物利用度， 可能能够为骨疾病中的sclerostin操作提供新的视角。

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.