Elucidation of the mechanisms of okadaic acid-induced apoptosis and cleavage of nucleolar proteins in osteoblasts

蛋白质磷酸化和去磷酸化已被认为是细胞功能的关键机制。冈田酸（OA）是一种有效的蛋白磷酸酶抑制剂，可诱导人成骨细胞凋亡。核因子κB （Nuclear factor -κB, NF-κB）是调控细胞生长和分化相关基因表达的重要转录因子。Western分析和k蛋白磷酸酶去磷酸化实验结果表明，OA处理人成骨MG63细胞可增强丝氨酸536上NF-κB的磷酸化水平。通过荧光素酶测定，OA刺激了MG63细胞中NF-κB的转录活性。这些结果表明，OA在MG63细胞中引起丝氨酸536上NF-κB的磷酸化，导致磷酸化NF-κB易位到细胞核，从而促进基因的转录活性。核仁蛋白是一种大量表达的核仁磷酸化蛋白，主要位于核仁中。培养细胞中核仁蛋白的染色模式与蛋白磷酸酶1δ相似。(PP1δ)。通过免疫细胞化学和免疫沉淀方法证实了核仁蛋白与人成骨细胞核仁中PP1δ的结合。OA处理的成骨细胞细胞核中agnor和核蛋白消失。在成骨细胞获得的蛋白质中检测到一个主要的条带，110 kDa。在凋亡的成骨细胞中，110 kDa蛋白的水平下降，而在oa诱导的凋亡成骨细胞中，该蛋白的水平增加了80 kDa。结果表明，PP18直接与成骨细胞核仁中的核仁蛋白结合，核仁蛋白在成骨细胞凋亡过程中发生断裂。

Protein phosphorylation and dephosphorylation has been recognized as a key mechanism in cell function. Okadaic acid (OA) is a potent inhibitor of protein phosphatases and induces apoptosis in human osteoblasts. Nuclear factor-kappa B (NF-κB) is an essential transcription factor in the control of expression of genes involved in cell growth and differentiation. Treatment of human osteoblastic MG63 cells with OA enhanced the phosphorylation level of NF-κB on serine 536, as judged from the results of Western analysis and a k protein phosphatase dephosphorylation assay. OA stimulated the transcriptional activity of NF-κB in MG63 cells by a luciferase assay. These findings indicate that OA elicit phosphorylation of NF-κB on serine 536 in MG63 cells, resulting in the translocation of phospho-NF-κB to the nucleus, thereby promoting transcriptional activity of genes.Nucleolin is an abundantly expressed nucleolar phosphoprotein and is located mainly in the nucleolus. The staining pattern of nucleolin in cultured cells is similar to that of protein phosphatase1δ. (PP1δ). Nucleolin was demonstrated to bind to PP1δ in nucleolus in human osteoblasts by using immunocytochemical and immunoprecipitation methods. AgNORs and nucleolin disappeared from the nuclei of the osteoblasts treated with OA. A major band, 110 kDa, was detected in the proteins obtained from osteoblastic cells. The level of the 110 kDa protein decreased in the apoptotic osteoblasts, whereas an additional band, 80 kDa, appeared and the level of this protein increased in the proteins prepared from OA-induced apoptotic osteoblasts. The present results indicate that PP18 directly binds to nucleolin in the nucleolus of osteoblasts and that nucleolin is cleaved during apoptosis in osteoblasts.