Heparan sulfate-dependent mechanisms of skeletogenesis

描述（由申请人提供）：颅面区域、躯干和四肢的骨骼发生受骨形态发生蛋白（BMP）、hedgehog和Wnt信号传导因子家族成员的调节。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外生骨疣（HME），我正在研究作为我正在进行的博士学位的一部分。论文工作。HME是一种儿科常染色体显性遗传疾病，在此期间，生长板附近的软骨膜内形成称为外生骨疣的带软骨帽的副产物，并导致生长迟缓、骨骼畸形、慢性疼痛和早发性骨关节炎。HME由编码负责HS合成的糖基转移酶的EXT 1和EXT 2中的杂合功能丧失突变引起。因此，患者表现出不同程度的HS缺乏，但尚不清楚HS缺乏如何导致外生骨疣形成和其他HME相关的骨骼病理学。为了阐明这些机制，我创建了条件小鼠胚胎突变体，其中Ext 1基因在软骨膜细胞中被消融。引人注目的是，这导致软骨膜内形成外生骨疣样软骨肿块，100%覆盖。在良好的相关性中，我发现在间充质细胞微团培养物中，HS缺乏显著增加了软骨形成分化和对促软骨形成因子如BMP 2的反应性。此外，其他人的研究表明，HS降解酶乙酰肝素酶（HPSE）在外生骨疣组织中的表达很高。因此，我的NRSA提案的中心假设是，HS是骨骼发生过程中抗软骨形成机制的主要调节剂。HS将通过促进抗软骨形成HS结合因子（包括转化生长因子2（TGF 2））的功能来这样做，所述抗软骨形成HS结合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，如在HME中所见。我的目标是：（i）分析HS对TGF 2和BMP在体内异位软骨形成中的作用的调节，并使用体外细胞系统来测试潜在的机制;和（ii）使用功能获得和丧失方法和细胞-蛋白质相互作用测定来确定乙酰肝素酶在修饰HS功能中的作用。拟议的研究将提供新的见解和更广泛的知识到HS的作用在发展中的骨骼和他们的错乱行动和功能HME。软骨再生在骨折修复过程中被重新激活，甚至在骨折的颅面骨中也经常通过软骨内骨化愈合;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在软骨和骨骼发生中的作用的研究具有广泛的生物学意义和转化医学的影响，我可以在未来的项目中追求。

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.