Proteoglycan Regulation of Growth Plate Cartilage Calcification

描述（由申请人提供）：软骨内骨化是一个非常复杂的过程，大量研究已经确定蛋白聚糖（PC）在该过程中对软骨钙化起关键作用。先前的工作已经证明，PG聚集体大小是钙化软骨中晶体大小和增殖的关键调节因子。此外，一些基因敲除动物模型表明，PG的合成，结构和降解与软骨内骨化过程中的软骨矿化密切相关。PG修饰也影响周围细胞因子的活性，如磷蛋白。然而，软骨钙化过程中PG形态的调节机制仍不清楚。因此，我们假设聚集蛋白聚糖修饰是生理性软骨钙化发生所必需的，并且蛋白聚糖形态和蛋白质活性之间存在微妙的平衡，最终导致软骨钙化。为了验证这些假设，我们建议使用小鼠间充质细胞系先前证明重演的过程中的endochondrialossification微培养。我们的具体目标是：1）确定生长板内聚集蛋白聚糖的周转机制。在软骨细胞成熟的不同阶段，微团系统将使用针对特定新表位开发的单克隆抗体进行探测，所述新表位由MMP、ADAMTS和半胱氨酸蛋白酶切割聚集蛋白聚糖产生。2)通过结合免疫阻断和RNA干扰敲减实验，对聚集蛋白聚糖分解蛋白酶进行研究，以验证聚集蛋白聚糖修饰是生理性软骨钙化发生所必需的假设。3)确定聚集蛋白聚糖硫酸化程度和类型与软骨钙化之间的关系。微团培养物将用氯酸钠（NaClO3）处理以抑制聚集蛋白聚糖的糖胺聚糖硫酸化，用软骨素酶处理以释放硫酸软骨素（CS），或用角蛋白酶处理以从基质释放硫酸角蛋白（KS）链。将评估这些处理组对聚集蛋白聚糖分解蛋白酶和磷蛋白活性的影响。软骨钙化异常的情况会影响新生儿和老年人的出生缺陷、生长畸形和遗传异常。为了预防和治疗这些疾病，必须了解生理性软骨钙化。这些研究的最终目的是利用细胞培养模型了解软骨内骨生长过程中生理性软骨钙化的机制。使用该信息，可以鉴定靶基因或生物分子，使得可以开发治疗软骨组织的功能性病症的临床解决方案。

DESCRIPTION (provided by applicant): Endochondral ossification is a very complex process and a great deal of research has determined that proteoglycans (PCs) have a critical role in cartilage calcification during this process. Previous work has demonstrated that PG aggregate size is a key regulator of crystal size and proliferation in calcified cartilage. Additionally, several knockout animal models have suggested that PG synthesis, structure, and degradation are intimately related to cartilage mineralization in the process of endochondral ossification. PG modification also influences the activity of surrounding cytokines, such as phosphoproteins. However, the mechanism by which PG morphology is regulated during cartilage calcification is still unknown. Thus we hypothesize that aggrecan modification is required for physiologic cartilage calcification to occur and that a delicate balance exists between proteoglycan morphology and protein activity ultimately resulting in cartilage calcification. To test these hypotheses, we propose to use a mouse mesenchymal cell line previously demonstrated to recapitulate the process of endochondral ossification in micromass culture. Our Specific Aims are to: 1) To determine the mechanism of aggrecan turnover within the growth plate. During different stages of chondrocyte maturation, the micromass system will be probed with monoclonal antibodies developed for specific neoepitopes that result from cleavage of aggrecan by MMPs, ADAMTSs, and cysteine proteases. 2) To test the hypothesis that aggrecan modification is required for physiologic cartilage calcification to occur using a combination of immune-blocking and RNAi knock down experiments for aggrecanolytic proteinases. 3) To determine the relationship between the degree and type of aggrecan sulfation and cartilage calcification. Micromass cultures will be treated with sodium chlorate (NaCIO3) to inhibit glycosaminoglycan sulfation of aggrecan, chondroitinase to release chondroitin sulfate (CS), or keratanase to release keratan sulfate (KS) chains from the matrix. The effects of these treatment groups on aggrecanolytic proteinase and phosphoprotein activity will be assessed. Conditions in which cartilage calcification is aberrant affect both the new born and the elderly in terms of birth defects, growth deformities, and inherited abnormalities. To prevent and treat these conditions it is essential to understand physiologic cartilage calcification. The ultimate goal of these studies is to understand the mechanisms involved in physiologic cartilage calcification during endochondral bone growth using a cell culture model. Using this information, target genes or biomolecules may be identified so that clinical solutions that treat the functional disorders of cartilage tissue may be developed.