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调节细胞增殖和分化中起着核心作用。这些活动之间的平衡似乎也可能在凋亡中起重要作用。冈田酸（OA）是一种有效的蛋白质磷酸酶型（PP-1）和2A型（PP-2A）的抑制剂，并增加了细胞蛋白的磷酸化，而不会激活蛋白激酶C. Wee证明，OA在SAOS-2细胞和DOSE依赖性的MG63细胞中均可诱导OA诱导OA诱导的蛋白质蛋白质。 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在10 nm OA的存在下，环己酰亚胺，放线霉素D或呼毒素。MG63细胞中有10 nm的OA.Cycloheximide，放线霉素D和嘌呤霉素在MG63细胞中显着保护了OA诱导的细胞毒性和细胞死亡。在OA存在的情况下，由HOECHST 33342检测的凋亡细胞数量也显着减少了这些试剂处理的MG63细胞的数量。此外，以剂量相关的方式，在用这些试剂处理的MG63细胞中，DNA梯形的形成强度降低。这些试剂在保护MG63细胞中保护OA诱导的凋亡方面的最大有效浓度与其他系统中有关THIR生物学作用的数据非常吻合。然而，环己酰亚胺，放线霉素D和嘌呤霉素并未预防由形态和生化技术确定的SAOS-2细胞中OA诱导的凋亡细胞死亡（Morimoto等人，在Press中）。