Proteoglycan Regulation of Growth Plate Cartilage Calcification

生长板软骨钙化的蛋白多糖调节

• 批准号: 7674929
• 负责人: Rhima Coleman
• 金额: $ 4.93万
• 依托单位: HOSPITAL FOR SPECIAL SURGERY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7674929
• 关键词: Achondroplasia; Affect; Animal Model; Behavior; Bone Development; Bone Growth; Calcified; Calpain; Cartilage; Cartilage injury; Cathepsins; Cell Culture Techniques; Cell Line; Cell Proliferation; Cells; Chondrocytes; Chondroitin Sulfates; Chondroitinases; Clinical; Complex; Congenital Abnormality; Cysteine Protease; Data; Deformity; Degenerative polyarthritis; Disintegrins; Dysplasia; Elderly; Epiphysial cartilage; Equilibrium; Fracture Healing; Functional disorder; Gene Targeting; Glycosaminoglycans; Goals; Growth; Hyaluronan; Hyaluronidase; Hypertrophy; Hypophosphatasia; Immune; In Vitro; Inherited; Keratan Sulfate; Knock-out; Laboratories; Length; Matrix Metalloproteinases; Mediating; Mesenchymal; Metalloproteases; Minerals; Modeling; Modification; Monoclonal Antibodies; Morphology; Mus; Nutritional; Osteocalcin; Pathway interactions; Pattern; Peptide Hydrolases; Phosphoproteins; Physiologic calcification; Physiological; Process; Proteinase 3; Proteins; Proteoglycan; RNA Interference; Regulation; Research; Rickets; Role; Site; Skeleton; Solutions; Staging; Structure; Syndrome; System; Term Birth; Testing; Thanatophoric Dysplasia; Time; Tissue Engineering; Tissues; Work; aggrecan; aggrecanase; bone; calcification; cathepsin K; chondrodysplasia; cytokine; keratan-sulfate endo-1,4-b-galactosidase; knock-down; knockout animal; link protein; long bone; mineralization; osteopontin; prevent; repaired; research study; sodium chlorate; sulfation; sulfotransferase; treatment effect

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.

描述（由申请人提供）：内侧软骨骨化是一个非常复杂的过程，大量研究确定蛋白聚糖（PC）在此过程中在软骨钙化中具有关键作用。先前的工作表明，PG聚集体大小是钙化软骨中晶体大小和增殖的关键调节剂。此外，几种敲除动物模型表明，在内侧软骨骨化过程中，PG合成，结构和降解与软骨矿化密切相关。 PG修饰还会影响周围细胞因子（例如磷蛋白）的活性。但是，在软骨钙化过程中调节PG形态的机制仍然未知。因此，我们假设生理软骨钙化需要摄取的修饰，并且蛋白聚糖形态与蛋白质活性之间存在微妙的平衡，最终导致软骨钙化。为了检验这些假设，我们建议使用先前证明的小鼠间充质细胞系，以概括微瘤培养中的内软骨骨化过程。我们的具体目的是：1）确定生长板中Aggrecan离职的机理。在软骨细胞成熟的不同阶段，将使用针对特定的新皮质的单克隆抗体进行探测，这些抗体是由MMPS，ADAMTSS和半胱氨酸蛋白酶裂解而导致的。 2）要测试假设，即生理软骨钙化需要进行脂肪蛋白的修饰，才能结合免疫阻滞和RNAi敲除脂肪蛋白溶解蛋白酶的实验。 3）确定脂肪蛋白硫酸盐和软骨钙化的程度和类型之间的关系。将用氯酸钠（NACIO3）处理微瘤培养物，以抑制脂肪蛋白酶的脂肪素硫酸盐硫酸盐硫酸盐硫酸盐酶释放硫酸软骨素（CS）或角膜酶以释放从基质中释放硫酸盐（KS）链的硫酸盐酶。这些治疗组对脂肪促蛋白酶和磷酸蛋白活性的影响将得到评估。软骨钙化异常的条件在出生缺陷，生长畸形和遗传异常方面都会影响新出生和老年人。为了预防和治疗这些疾病，必须了解生理软骨钙化。这些研究的最终目的是了解使用细胞培养模型在软骨骨生长过程中生理软骨钙化所涉及的机制。使用这些信息，可以鉴定靶基因或生物分子，以便可以开发出处理软骨组织功能障碍的临床溶液。