EFFECTS OF MUTATIONS IN COLLAGEN GENES ON CARTILAGE MATRIX

本项目是前一个项目的项目3和项目4的整合。该项目的主要目标是建立对两种软骨特异性胶原蛋白基因（COL2A和COL9A2）的不同突变如何导致软骨发育不良和过早OA的详细机制理解。目的是利用体外和体内系统来充分了解这些突变对软骨基质的生物化学和结构、胚胎骨骼和关节发育以及最终对成人关节软骨的影响。为了实现这一目标，我们将利用我们在之前的资助期间开发的聚2-羟乙基甲基丙烯酸酯（poly- hema）软骨细胞培养系统，该系统可以长期保存软骨细胞的软骨特异性表型。其次，我们将建立敲入小鼠系，这些敲入小鼠在软骨特异性胶原蛋白基因中具有导致人类软骨发育不良和过早OA的相同突变。第三，我们将创建转基因小鼠系，其中突变的胶原蛋白分子可以以软骨特异性和有条件的方式表达。我们将使用形态学、组织学和组织病理学评估来详细检查所得到的动物系，并将提供给我们的合作者和其他研究者来研究突变在体内的功能影响。此外，这些小鼠的关节软骨将采用高度敏感的渗透加载方法进行生物力学检查，以回答突变对组织生物材料特性的功能影响，并试图建立可能与骨关节炎发病机制直接相关的结构/功能关系。第四，我们将利用纯化的胶原分子和体外原纤维形成和蛋白质相互作用的实验来更好地了解这些突变对胶原单体结构、原纤维组装和结构以及胶原II和IX之间的分子相互作用的影响。本项目的总体目标将通过追求以下具体目标来实现：(1)确定突变的细胞外基质基因表达对培养软骨细胞表型的影响以及由此产生的软骨基质的功能特征；(2)通过在相应的小鼠基因中引入（敲入）类似的人类突变，生成由人类COL2A1和COL9A2基因突变引起的人类发育不良小鼠模型；(3)确定突变型胶原在成年小鼠关节中条件组织特异性表达的影响；(4)研究Arg75、Arg519和Arg789对II型胶原中Cys突变对胶原原纤维组装的影响，研究野生型和突变型的II型胶原和IX型胶原之间的相互作用。

This project is a consolidation of Project 3 and Project 4 of the previous Program Project. The major goal of this project will be to establish a detailed mechanistic understanding of how different mutations in two cartilage-specific collagen genes (COL2A and COL9A2) result in chondrodysplasia and premature OA. The aim will be to utilize in vitro and in vivo systems to gain a full understanding of the effects of these mutations on the biochemistry and structure of the cartilage matrix, on embryonic skeletal and joint development and finally on adult articular cartilage. To achieve this, we will exploit the poly 2-hydroxyethyl methacrylate (poly-HEMA) chondrocyte culture system we developed during the previous funding periods which allows the long-term preservation of the cartilage-specific phenotype of chondrocytes. Second, we will establish lines of knock-in mice that harbor the same mutations in cartilage-specific collagen genes that cause chondrodysplasia and premature OA in humans. Third, we will create transgenic mouse lines in which the mutated collagen molecules can be expressed in a cartilage specific and conditional manner. The resulting animal lines will be examined in detail employing morphologic, histologic, and histopathological assessments and will be made available to our collaborators and to other investigators to study the functional effects of the mutations in vivo. Furthermore, articular cartilage from these mice will be examined biomechanically employing a highly sensitive osmotic loading method to answer the functional impact of the mutations on the biomaterial properties of the tissue and attempt to establish structure/function relationships that may be of direct relevance to the pathogenesis of osteoarthritis. Fourth, we will utilize purified collagen molecules and in vitro fibril formation and protein interaction assays to obtain a better understanding of the effects of these mutations on collagen monomer structure, fibril assembly and structure, and molecular interactions between collagens II and IX. The overall goal of this project will be achieved by pursuing the following Specific Aims: (1) To determine the effects of expression of mutated extracellular matrix genes on the phenotype of chondrocytes in culture and the functional characteristics of the resulting cartilaginous matrix; (2) To generate authentic mouse models of the human chrondrodysplasias caused by mutations in the human COL2A1 and COL9A2 genes by introducing (knocking-in) the analogous human mutations in the corresponding mouse genes; (3) To determine the effects of conditional tissue-specific expression of mutant collagens in the articular of adult mice; and (4) To study the effects of Arg75, Arg519 and Arg789 to Cys mutations in type II collagen on collagen fibril assembly and to study interactions between wild type and mutant collagen II and collagen IX.