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

蛋白质的磷酸化和去磷酸化被认为是细胞功能中的一个关键机制。冈田酸是一种有效的蛋白磷酸酶抑制剂，可诱导人成骨细胞的凋亡。核因子-kappaB(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a-k蛋白磷酸酶去磷酸化实验结果显示，人成骨细胞MG63细胞经OA处理后，丝氨酸536上的核因子-κB的磷酸化水平增加。荧光素酶活性测定显示，OA可刺激MG6 3细胞中NF-κB的转录活性。这些结果表明，在MG63细胞中，OA诱导丝氨酸536上的NF-κB的磷酸化，导致磷酸化的NF-κB移位到细胞核，从而促进基因的转录活性。核仁蛋白是一种丰富表达的核仁磷酸蛋白，主要位于核仁。核仁在培养细胞中的染色模式与蛋白磷酸酶1δ的染色模式相似。(pp1δ)。用免疫细胞化学和免疫沉淀法证明核素能与人成骨细胞核仁中的Pp1δ结合。成骨细胞经OA处理后，核仁组成区和核仁核仁消失。在从成骨细胞获得的蛋白质中检测到一条110 kDa的主带。在凋亡的成骨细胞中，110 kDa蛋白的表达水平降低，而在OA诱导的成骨细胞中，该蛋白的表达水平增加，并出现一条80 kDa的额外条带。目前的结果表明，PP18直接与成骨细胞核仁中的核仁结合，核仁在成骨细胞凋亡过程中发生裂解。

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.