Protein Dephosphorylation and Apoptosis in Osteoblast

成骨细胞中的蛋白质去磷酸化和细胞凋亡

基本信息

批准号：
09671860
负责人：
HANEJI Tatsuji
金额：
$ 1.92万
依托单位：
Kyushu Dental College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671860/
关键词：
osteoblast apoptosis protein dephosphorylation okadaic acid calyculin A osteogenesis Saos-2 MG63 カリクレインA Saos-1 細胞分化

项目摘要

Phosphorylation and dephosphorylation play a central role in the regulation of cellular proliferation and differentiation. It seems likely that the balance between these activities also plays an important parts in apoptosis. Okadaic acid (OA) is a potent inhibitor of protein phosphatases type 1 (PP-1) and type 2A (PP-2A) and increases the phosphorylation of cellular proteins without activation of protein kinase C.We demonstrated that OA induces apoptosis in both Saos-2 cells and MG63 cells in a dose-dependent manner with a maximum effective concentration of 10 nM (Morimoto et al, 1997). Because inhibition of protein phosphatase activity by OA may be a general way of triggering apoptosis in some kinds of cells, it is believed that new protein synthesis is not required for OA-induced apoptosis.To determine if inhibition of the protein synthesis or RNA synthesis could protect against the OA-induced apoptosis in osteoblasts, we cultured the cells in media containing varying concentrations of cycloheximide, actinomycin D, or puromycin in the presence of 10 nM OA.Cycloheximide, actinomycin D, and puromycin significantly protected against OA-induced cytotoxicity and cell death in MG63 cells. The number of apoptotic cells examined by the Hoechst 33342 staining also significantly decreased in MG63 cells treated with these reagents in the presence of OA.Moreover, the intensity of DNA ladder formation was decreased in MG63 cells treated with these reagents in a dose-dependent manner. The maximum effective concentrations of these reagents on the protection of OA-induced apoptosis in MG63 cells are in good agreement with the data concerning thier biological effects in other systems. However, cycloheximide, actinomycin D, and puromycin did not protect against the OA-induced apoptotic cell death in Saos-2 cells determined by the morphological and biochemical techniques (Morimoto et al, in press).

磷酸化和去磷酸化在细胞增殖和分化的调节中起着核心作用。这些活动之间的平衡似乎也在细胞凋亡中发挥着重要作用。冈田酸 (OA) 是 1 型蛋白磷酸酶 (PP-1) 和 2A 型蛋白磷酸酶 (PP-2A) 的有效抑制剂，可在不激活蛋白激酶 C 的情况下增加细胞蛋白的磷酸化。我们证明 OA 会诱导 Saos 细胞凋亡-2 细胞和 MG63 细胞以剂量依赖性方式作用，最大有效浓度为 10 nM (Morimoto 等，1997)。由于OA对蛋白磷酸酶活性的抑制可能是引发某些细胞凋亡的一般方式，因此认为OA诱导的细胞凋亡不需要新的蛋白质合成。确定抑制蛋白质合成或RNA合成是否可以为了防止 OA 诱导的成骨细胞凋亡，我们在含有不同浓度的放线菌酮、放线菌素 D 或嘌呤霉素的培养基中培养细胞，并在 10 nM OA 存在下进行培养。放线菌素 D 和嘌呤霉素可显着防止 MG63 细胞中 OA 诱导的细胞毒性和细胞死亡。在 OA 存在的情况下，用这些试剂处理的 MG63 细胞中，通过 Hoechst 33342 染色检查的凋亡细胞数量也显着减少。此外，用这些试剂处理的 MG63 细胞中 DNA 阶梯形成的强度以剂量依赖性方式降低。。这些试剂对保护 OA 诱导的 MG63 细胞凋亡的最大有效浓度与其他系统中有关其生物学效应的数据非常一致。然而，通过形态学和生化技术测定，放线菌酮、放线菌素 D 和嘌呤霉素不能防止 Saos-2 细胞中 OA 诱导的细胞凋亡（Morimoto 等人，出版中）。