Heparan sulfate-dependent mechanisms of skeletogenesis

描述（由申请人提供）：颅面区、躯干和四肢的骨骼发生受骨形态发生蛋白（BMP）、hedgehog和Wnt信号因子家族成员的调控。有趣的是，这些因子都是硫酸肝素（HS）结合蛋白，研究表明HS链影响其在靶细胞上的分布、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，我正在进行的博士论文工作的一部分研究的遗传性多发性外生骨病（HME）就是一个很好的例子。HME是一种小儿常染色体显性遗传病，在这种疾病中，生长板旁边的软骨膜内形成软骨覆盖的外生骨瘤，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HS合成的糖基转移酶的EXT1和EXT2的杂合性功能缺失突变引起的。因此，患者表现出不同程度的HS缺乏症，但尚不清楚HS缺乏症如何导致外生骨骺形成和其他与hme相关的骨骼病变。为了阐明这些机制，我创建了条件小鼠胚胎突变体，其中在软骨膜细胞中切除了Ext1基因。引人注目的是，这导致硬骨膜内形成外生性软骨样肿块，其外显率为100%。我在间充质细胞微团培养中发现，HS缺乏显著增加了软骨分化和对BMP2等促软骨因子的反应。此外，其他研究表明，hs降解酶肝素酶（HPSE）在外生组织中表达高。因此，我的NRSA提案的中心假设是HS是骨骼形成过程中抗软骨形成机制的主要调节因子。HS可通过促进抗软骨生成HS结合因子（包括转化生长因子2 (TGF2)）的功能来达到这一目的，这些因子通常需要调节软骨膜表型和软骨-软骨膜相互作用。由于HS缺乏导致的这种机制的先天性改变会导致异位软骨形成，例如HME。我的目标是：(i)分析HS在体内异位软骨形成中对TGF2和BMP作用的调节，并利用体外细胞系统测试其潜在机制；和（ii）确定肝素酶在改变HS功能中的作用，使用功能获得和功能丧失方法和细胞-蛋白质相互作用测定。所提出的研究将为hhs在骨骼发育中的作用及其在HME中的紊乱行为和功能提供新的见解和更广泛的知识。在骨折修复过程中，甚至在通常通过软骨内成骨愈合的骨折颅面骨中，软骨形成被重新激活；它也正在生物工程方法中进行实验测试，以修复和再生颅面和肢体骨骼结构。因此，我提出的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进行。

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.