Proteoglycan Regulation of Growth Plate Cartilage Calcification

描述(申请人提供)：软骨内骨化是一个非常复杂的过程，大量研究已确定蛋白多糖(PC)在这一过程中对软骨钙化起关键作用。先前的工作已经证明PG聚集体的大小是钙化软骨中晶体大小和增殖的关键调节因素。此外，一些基因敲除的动物模型表明，PG的合成、结构和降解与软骨内成骨过程中的软骨矿化密切相关。PG修饰也会影响周围细胞因子的活性，如磷蛋白。然而，PG形态在软骨钙化过程中的调控机制尚不清楚。因此，我们假设生理性软骨钙化需要蛋白多糖修饰，蛋白多糖形态和蛋白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，我们建议使用先前演示的小鼠间充质细胞系来概括在微团培养中软骨内成骨的过程。我们的具体目标是：1)确定aggrecan在生长板内周转的机制。在软骨细胞成熟的不同阶段，将用针对特定新表位的单抗来探索微质量系统，这些新表位是由MMPs、ADAMTS和半胱氨酸蛋白酶切割聚集素而产生的。2)结合免疫阻断和RNA干扰抑制蛋白水解酶实验，验证生理性软骨钙化需要aggrecan修饰的假设。3)探讨聚集素硫酸盐化的程度和类型与软骨钙化的关系。用氯酸钠(NaClO_3)处理微团培养物以抑制聚集素的糖胺多聚糖硫酸化，用软骨素酶从基质中释放硫酸软骨素(CS)，或用角聚糖酶(KS)从基质中释放角蛋白(KS)链。将评估这些处理组对抗凝蛋白水解酶和磷酸蛋白活性的影响。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情况，了解生理性软骨钙化是必不可少的。这些研究的最终目的是通过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。利用这些信息，可以识别靶基因或生物分子，从而开发出治疗软骨组织功能障碍的临床解决方案。

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.