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首页> 外文期刊>Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research >MDM2 antagonist Nutlin-3 suppresses the proliferation and differentiation of human pre-osteoclasts through a p53-dependent pathway.
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MDM2 antagonist Nutlin-3 suppresses the proliferation and differentiation of human pre-osteoclasts through a p53-dependent pathway.

机译：MDM2拮抗剂Nutlin-3通过p53依赖性途径抑制人类破骨细胞的增殖和分化。

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摘要

Exposure of human pre-osteoclasts to the MDM2 antagonist Nutlin-3 activated the p53 pathway and significantly decreased the entry of pre-osteoclasts in the S phase in response to RANKL. Moreover, repeated exposure to Nutlin-3 suppressed osteoclastic differentiation, without affecting cell survival at any culture time. INTRODUCTION: The p53 oncosuppressor coordinates an intracellular network involved in protection from malignant transformation and cell cycle control; its activation is tightly regulated by the murine double minute 2 (MDM2) gene and p53-MDM2 interaction can be disrupted by selective small molecule inhibitors, the Nutlins. Although the ability of Nutlins to suppress the growth of wildtype p53 tumors has been clearly established, their biological activity in normal cells and tissues has not been extensively studied. MATERIALS AND METHODS: Peripheral blood mononuclear cell pre-osteoclasts were cultured with macrophage-colony stimulating factor (M-CSF) + RANKL or co-cultured with SaOS-2 osteosarcoma cells in the presence of IL-1beta to induce osteoclastic differentiation. Cell cycle was analyzed by BrdU incorporation. The degree of osteoclastic differentiation was monitored at different culture times by TRACP and DAPI staining, as well as by TRACP-5b ELISA. Finally, the role of p53 in mediating the biological activity of Nutlin-3 was studied using specific siRNA. RESULTS: Exposure of human pre-osteoclasts to RANKL induced an early (24 h) increase in the percentage of cells in the S phase, followed by the exit from the cell cycle at later time-points. The simultaneous addition of Nutlin-3 and RANKL dose-dependently decreased the percentage of pre-osteoclasts in the S phase and induced a rapid accumulation of p53 protein coupled with the induction of p53 target genes. Unexpectedly, the administration of Nutlin-3 to pre-osteoclasts at early culture times significantly suppressed the final output of osteoclasts at day 14 of culture. The role of p53 in mediating this biological activity of Nutlin-3 was underscored by gene knockdown experiments, in which the anti-osteoclastic activity of Nutlin-3 was significantly counteracted by siRNA specific for p53. Nutlin-3 also significantly decreased the formation of osteoclasts in a co-culture system of SaOS-2 osteosarcoma and pre-osteoclastic cells. CONCLUSIONS: These findings indicate that Nutlin-3 abrogates both pre-osteoclastic proliferation and differentiation through a p53-dependent pathway and may have therapeutic implications for those neoplastic diseases characterized by an abnormal osteoclastic activity.
机译：将人破骨细胞暴露于MDM2拮抗剂Nutlin-3激活了p53途径，并显着减少了响应RANKL的破骨细胞进入S期。此外，反复暴露于Nutlin-3可以抑制破骨细胞分化，而不会影响任何培养时间的细胞存活。简介：p53抑癌药可协调参与保护细胞免受恶性转化和细胞周期控制的细胞内网络。它的激活受到鼠双分钟2（MDM2）基因的严格调控，而p53-MDM2的相互作用可被选择性的小分子抑制剂Nutlins破坏。尽管已经明确建立了坚果蛋白抑制野生型p53肿瘤生长的能力，但尚未广泛研究它们在正常细胞和组织中的生物学活性。材料与方法：外周血单核细胞破骨细胞与巨噬细胞集落刺激因子（M-CSF）+ RANKL一起培养，或在IL-1β存在下与SaOS-2骨肉瘤细胞共培养以诱导破骨细胞分化。通过BrdU掺入分析细胞周期。通过TRACP和DAPI染色以及TRACP-5b ELISA监测不同培养时间的破骨细胞分化程度。最后，使用特异性siRNA研究了p53在调节Nutlin-3生物学活性中的作用。结果：人类破骨细胞暴露于RANKL导致S期细胞百分比提前（24小时）增加，随后在随后的时间点退出细胞周期。同时添加Nutlin-3和RANKL剂量依赖性地降低了S期破骨细胞的百分率，并诱导了p53蛋白的快速积累以及p53靶基因的诱导。出乎意料的是，在培养的早期，对破骨细胞前体给予Nutlin-3会显着抑制培养第14天的破骨细胞最终产量。基因敲低实验强调了p53在介导Nutlin-3生物学活性中的作用，其中Nutlin-3的抗破骨细胞活性被p53特异的siRNA显着抵消。在SaOS-2骨肉瘤和破骨前细胞的共培养系统中，Nutlin-3还显着减少了破骨细胞的形成。结论：这些发现表明，Nutlin-3通过p53依赖性途径消除了破骨前的增殖和分化，并且可能对那些以破骨活性异常为特征的肿瘤疾病具有治疗意义。

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