Proteoglycan Regulation of Growth Plate Cartilage Calcification

描述（由申请人提供）：软骨内成骨是一个非常复杂的过程，大量研究已经确定，在这一过程中，蛋白聚糖（PCs）在软骨钙化中起着关键作用。先前的研究表明PG聚集体大小是钙化软骨晶体大小和增殖的关键调节因子。此外，一些敲除动物模型表明，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修饰也影响周围细胞因子的活性，如磷酸化蛋白。然而，在软骨钙化过程中PG形态调控的机制尚不清楚。因此，我们假设聚集蛋白修饰是发生生理性软骨钙化所必需的，并且在蛋白聚糖形态和最终导致软骨钙化的蛋白质活性之间存在微妙的平衡。为了验证这些假设，我们建议使用先前证明的小鼠间充质细胞系在微团培养中再现软骨内成骨的过程。我们的具体目标是：1)确定生长板内聚集蛋白周转的机制。在软骨细胞成熟的不同阶段，将用针对特定新表位的单克隆抗体探测微团系统，这些新表位是由MMPs、ADAMTSs和半胱氨酸蛋白酶切割聚集蛋白产生的。2)利用免疫阻断和RNAi敲除聚合酶实验相结合的方法，验证生理性软骨钙化需要聚合蛋白修饰的假设。3)确定聚集蛋白硫酸化程度和类型与软骨钙化的关系。用氯酸钠（NaCIO3）处理微块培养物，以抑制聚集蛋白的糖胺聚糖硫酸化，抑制软骨素酶释放硫酸软骨素（CS），或抑制角朊酶从基质中释放硫酸角朊（KS）链。将评估这些治疗组对聚解蛋白酶和磷蛋白活性的影响。在软骨钙化异常的情况下，新生儿和老年人在出生缺陷、生长畸形和遗传异常方面都受到影响。为了预防和治疗这些疾病，了解生理性软骨钙化是至关重要的。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，可以确定靶基因或生物分子，从而可以开发出治疗软骨组织功能紊乱的临床解决方案。

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.