Heparan sulfate-dependent mechanisms of skeletogenesis

描述(申请人提供)：颅面部、躯干和四肢的骨骼形成由骨形态发生蛋白(BMP)、Hedgehog和Wnt信号因子家族成员调控。有趣的是，这些因子都是硫酸乙酰肝素(HS)结合蛋白，研究表明HS链影响它们在靶细胞上的分布、生物利用度和功能。许多先天性疾病是由HS相关机制的突变引起的，一个恰当的例子是遗传性多发性骨软骨病(HME)，这是我正在进行的博士论文工作的一部分。HME是一种儿童常染色体显性遗传性疾病，在此期间，在生长板旁边的软骨膜内形成被称为外骨瘤的软骨覆盖的突起，导致生长迟缓、骨骼畸形、慢性疼痛和早发性骨关节炎。HME是由编码负责HS合成的糖基转移酶的EXT1和EXT2杂合性功能丧失突变引起的。因此，患者表现出不同程度的HS缺乏症，但尚不清楚HS缺乏症是如何导致外生骨疣形成和其他HME相关的骨骼病理的。为了阐明这些机制，我创造了有条件的小鼠胚胎突变体，其中Ext1基因在软骨膜细胞中被去除。值得注意的是，这导致在软骨膜内形成100%外露的外生骨软骨瘤样软骨团块。在良好的相关性中，我发现在间充质细胞微团培养中，HS缺乏显著增加了软骨分化和对促软骨因子如BMP2的反应性。另外，前人的研究表明，HS降解酶乙酰肝素酶(HPSE)在骨软骨瘤组织中的表达水平较高。因此，我的NRSA建议的中心假设是HS是骨骼形成过程中抗软骨形成机制的主要调节因子。HS将通过促进包括转化生长因子2(TGF2)在内的抗软骨性HS结合因子的功能来实现这一点，这些因子通常是调节软骨膜表型和软骨-软骨膜相互作用所必需的。由于HS缺乏导致的这种机制的先天改变将导致异位软骨生成，如HME中所见。我的目标是：(I)分析HS对TGF2和BMP在体内异位软骨形成中的作用，并使用体外细胞系统测试潜在的机制；以及(Ii)利用功能获得和丧失方法以及细胞-蛋白质相互作用分析，确定肝素酶在改变HS功能中的作用。拟议的研究将为HS在发育中的骨架中的作用以及它们在HME中的错乱行为和功能提供新的见解和更广泛的知识。软骨生成在骨折修复期间重新激活，甚至在骨折的头面部骨骼中也会重新激活，这些骨骼通常通过软骨内骨化愈合；它也正在通过生物工程方法进行实验测试，以修复和再生颅面和四肢骨骼结构。因此，我提出的关于HS在软骨形成和骨骼形成中的作用的研究具有广泛的生物学意义和转化医学意义，我可以在未来的项目中继续研究。 公共卫生相关性：骨骼发育和生长是基本的过程，如果有缺陷，可能会导致头面部、躯干和四肢骨骼的主要病理变化，并可能导致身体功能和生活质量的损害。这笔资金将使我能够完成生理性骨骼发生和一种叫做遗传性多发性骨软骨病的不正常形式的博士论文工作。该项目将提供对机制的新见解，从而提出修复和再生有缺陷的骨骼元素和恢复功能的可能治疗方法。

DESCRIPTION (provided by applicant): Skeletogenesis in the craniofacial region, trunk and limbs is regulated by members of the bone morphogenetic protein (BMP), hedgehog and Wnt families of signaling factors. Interestingly, these factors are all heparan sulfate (HS)-binding proteins, and studies have shown that the HS chains influence their distribution and bioavailability and function on target cells. A number of congenital conditions are caused by mutations in HS- related mechanisms, and a case in point is Hereditary Multiple Exostoses (HME) that I am studying as part of my ongoing Ph.D. thesis work. HME is a pediatric autosomal dominant disorder during which cartilage-capped outgrowths called exostoses form within perichondrium next to the growth plate and cause growth retardation, skeletal deformities, chronic pain and early onset osteoarthritis. HME is caused by heterozygous loss-of- function mutations in EXT1 and EXT2 that encode glycosyltransferases responsible for HS synthesis. Thus, patients display varying degrees of HS deficiency, but it is unclear how HS deficiency leads to exostosis formation and other HME-associated skeletal pathologies. To clarify these mechanisms, I created conditional mouse embryo mutants in which the Ext1 gene was ablated in perichondrial cells. Strikingly, this caused formation of exostosis-like cartilaginous masses within perichondrium with 100% penetrance. In good correlation, I found in mesenchymal cell micromass cultures that HS deficiency markedly increased chondrogenic differentiation and responsiveness to pro-chondrogenic factors such as BMP2. Additionally, studies by others showed that expression of HS-degrading enzyme heparanase (HPSE) is high in exostosis tissue. Thus, the central hypothesis of my NRSA proposal is that HS is a major regulator of anti- chondrogenic mechanisms during skeletogenesis. HS would do so by promoting the function of anti- chondrogenic HS-binding factors including transforming growth factor 2 (TGF2) that are normally needed to regulate the perichondrial phenotype and chondro-perichondrial interactions. Congenital alterations in such mechanisms due to HS deficiency would cause ectopic chondrogenesis such as that seen in HME. My Aims are: (i) To analyze HS regulation of TGF2 and BMP action in ectopic cartilage formation in vivo and use in vitro cell systems to test underlying mechanisms; and (ii) To determine the roles of heparanases in modifying HS function, using gain- and loss-of-function approaches and cell-protein interaction assays. The proposed studies will provide novel insights and a wider breadth of knowledge into HS roles in the developing skeleton and their deranged action and function in HME. Chondrogenesis is reactivated during fracture repair and even in fractured craniofacial bones that often heal by endochondral ossification; it is also being experimentally tested in bioengineering approaches to repair and regenerate craniofacial and limb skeletal structures. Thus, my proposed studies on HS roles in chondrogenesis and skeletogenesis have broad biological significance and translational medicine implications that I could pursue in future projects. PUBLIC HEALTH RELEVANCE: Skeletal development and growth are fundamental processes that when defective, can cause major pathologies in the craniofacial, trunk and limb skeleton and can lead to impairment of body function and quality of life. This grant will allow me to complete my Ph.D. thesis work on physiologic skeletogenesis and an abnormal form of it called Hereditary Multiple Exostoses. The project will provide novel insights into mechanisms and will thus suggest possible therapeutic ways to repair and regenerate defective skeletal elements and restore function.