Comparison of Mineralizing Synchronous UMR106-01 cells with UMR106-01 cells treated with Mineralization Inhibitor AEBSF

UMR106-01 osteoblastic cells are a model for studying bone mineralization. We have shown that mineralization is temporally synchronized within cultures grown under defined conditions . Cells are plated at time zero and differentiate into osteoblastic phenotype by 64 h later. If an exogenous phosphate source is added to the cultures, the cells form and deposit hydroxyapatite mineral within distinct extracellular supramolecular lipid protein complexes termed biomineralization foci (BMF) starting 12 h later. Mineralization is largely complete by 24 h later (88 h after plating). We have also shown that AEBSF, covalent serine protease inhibitor, blocks mineralization within BMF and inhibits the fragmentation of several proteins related to biomineralization. The present experiment was designed to test whether AEBSF treatment for 12 h has an effect on transcription by UMR106-01 osteoblastic cells. AEBSF is known to inactivate several serine proteases including SKI-1 (site 1, subtilisin kexin protease-1).SKI-1 functions intracellularly to activate transmembrane bound transcription factor precursors releasing the transcriptionally active N-terminal portions to imported into the nucleus. Thus, if AEBSF blocks transcription of mineralization related genes, it would support a role for SKI-1 in gene regulation in mineralizing UMR106-01 osteoblastic cells.UMR106-01成骨细胞（UMR106-01 osteoblastic cells）是研究骨矿化（bone mineralization）的经典细胞模型。课题组前期研究证实，在限定培养条件下，该细胞培养物内的矿化过程呈现严格的时间同步性。实验于0时刻接种细胞，培养64小时后细胞可分化为成骨表型。若向培养液中添加外源性磷酸盐源，细胞将在12小时后，于被命名为生物矿化焦点（biomineralization foci, BMF）的特异性细胞外超分子脂质蛋白复合物内形成并沉积羟基磷灰石（hydroxyapatite）矿物。至接种后88小时（即添加磷酸盐源后24小时），矿化过程基本完成。本团队此前还证实，共价丝氨酸蛋白酶抑制剂AEBSF可阻断BMF内部的矿化过程，并抑制多种与生物矿化相关的蛋白质发生降解片段化。本实验旨在探究12小时的AEBSF处理是否会对UMR106-01成骨细胞的转录过程产生影响。已知AEBSF可灭活包括SKI-1（site 1, subtilisin kexin protease-1）在内的多种丝氨酸蛋白酶。SKI-1在细胞内发挥功能，可激活跨膜结合的转录因子前体，将具有转录活性的N端结构释放入细胞核。因此，若AEBSF能够阻断矿化相关基因的转录，则可佐证SKI-1在矿化的UMR106-01成骨细胞的基因调控中发挥作用。