Protein Dephosphorylation and Apoptosis in Osteoblast

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在细胞凋亡中起着重要作用。冈田酸是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，可以增加细胞蛋白的磷酸化，而不激活蛋白激酶C。研究表明，冈田酸以剂量依赖的方式诱导Saos-2细胞和MG63细胞凋亡，最大有效浓度为10 nM(Morimoto et al，1997)。为了确定抑制蛋白质合成或RNA合成是否能对抗OA诱导的成骨细胞的凋亡，我们将细胞培养在含有不同浓度的放线菌素、放线菌素D或嘌呤霉素的培养液中。Hoechst 33342染色显示，在OA存在的情况下，这些试剂处理的MG63细胞的凋亡细胞数也显著减少，并且这些试剂处理的MG63细胞的DNA梯带形成强度也以剂量依赖的方式减少。这些试剂对OA诱导的MG63细胞凋亡的保护作用的最大有效浓度与其他系统中有关其生物学效应的数据是一致的。然而，放线菌素D、放线菌素D和腐霉素A并不能通过形态和生物化学技术来确定其对OA诱导的Saos-2细胞凋亡的保护作用(Morimoto等人，在出版社中)。

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).