Quantitative and qualitative control of hyaluronan which plays a role in the assembly of extracellular matrix as a backbone of proteoglycan aggregate

本项目的目的是1)阐明影响透明质酸解聚酶的因素，该酶能将高相对分子质量的透明质酸降解成相对分子质量为40,000的片段；2)在透明质酸合成酶抑制剂4-甲基贝母内酯的存在下，培养细胞形成缺乏透明质酸的细胞外基质。首先，肝素酶处理培养的成纤维细胞细胞层可以刺激外源透明质酸的解聚。肝素酶处理也表现出对高相对分子质量透明质酸合成的抑制。这些结果提示，细胞表面硫酸乙酰肝素可能影响透明质酸代谢。其次，对细胞在0.5 mM 4-甲基伞形酮存在下培养形成的透明质酸缺乏的细胞外基质进行分析，发现细胞层中CD44和纤维连接蛋白的表达减少。细胞层中I型胶原含量无明显变化，而4-甲基伞形酮可使细胞内I型胶原分子含量增加。这些现象可能是由于细胞外基质中缺乏透明质酸所致。此外，还探讨了透明质酸与基质金属蛋白酶的关系。在外源性透明质酸存在下培养细胞，用酶谱和免疫印迹法比较细胞中基质金属蛋白酶的活性。结果发现，高浓度(1 mg/ml以上)的高分子量透明质酸对基质分解素-1的分泌有较强的抑制作用。

The purposes of this project are 1) to clarify the factors that affect hyaluronan-depolymerizing enzyme which degrade high-molecular-weight hyaluronan into fragments with molecular weight of 40,000, and 2) to characterize the hyaluronan-deficient extracellular matrix which were formed by cultivating tcells in the presence of 4-methylimbelliferone, an inhibitor of hyaluronan synthase. First, heparitinase treatment of the cell layer of cultured fibroblasts resulted the stimulation of the depolymerization of exogenous hyaluronan. Heparitinase treatment also showed the inhibition of the synthesis of high-molecular-weight hyaluronan. These results suggest that the cell surface heparan sulfate may affect hyaluronan metabolisim. Second, the analysis of hyaluronan-deficient extracellular matrix, which can be formed by cultivation of the cells in the presence of 0.5 mM 4-methylumbelliferone, showed the decrease of CD44 and fibronectin in cell layer. The amount of type I collagen in cell layer was not changed, while an increase of the molecule was observed in the medium by 4-methylumbelliferone. These phenomena may be caused by the lack of hyaluronan in extracellular matrix. Furthermore, the relation between hyaluronan and matrix metalloproteinases were investigated. Cells were cultured in the presence of exogenous hyaluronan and the activities of matrix metalloproteinases were compared with that of control cells by zymography and western blotting. As a result, the excretion of stromelysin-1 was found to be strongly suppressed by high-molecular weight hyaluronan with high-concentration of more than 1 mg/ml.