Zauli Giorgio, Rimondi Erika, Corallini Federica, Fadda Roberto, Capitani Silvano, Secchiero Paola
Department of Biomedicine, University of Trieste, Trieste, Italy.
J Bone Miner Res. 2007 Oct;22(10):1621-30. doi: 10.1359/jbmr.070618.
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.
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.
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.
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.
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.
将人破骨细胞前体细胞暴露于MDM2拮抗剂Nutlin-3可激活p53信号通路，并显著减少破骨细胞前体细胞在RANKL刺激下进入S期。此外，反复暴露于Nutlin-3可抑制破骨细胞分化，且在任何培养时间均不影响细胞存活。
p53肿瘤抑制因子协调一个参与防止恶性转化和细胞周期控制的细胞内网络；其激活受到小鼠双微体2（MDM2）基因的严格调控，p53与MDM2的相互作用可被选择性小分子抑制剂Nutlins破坏。尽管Nutlins抑制野生型p53肿瘤生长的能力已得到明确证实，但其在正常细胞和组织中的生物学活性尚未得到广泛研究。
外周血单核细胞破骨细胞前体细胞与巨噬细胞集落刺激因子（M-CSF）+RANKL一起培养，或在IL-1β存在的情况下与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。在不同培养时间通过TRACP和DAPI染色以及TRACP-5b ELISA监测破骨细胞分化程度。最后，使用特异性siRNA研究p53在介导Nutlin-3生物学活性中的作用。
将人破骨细胞前体细胞暴露于RANKL可诱导早期（24小时）S期细胞百分比增加，随后在后期时间点退出细胞周期。同时添加Nutlin-3和RANKL可剂量依赖性地降低破骨细胞前体细胞在S期的百分比，并诱导p53蛋白快速积累以及p53靶基因的诱导。出乎意料的是，在培养早期将Nutlin-3给予破骨细胞前体细胞可显著抑制培养第14天破骨细胞的最终产量。基因敲低实验强调了p53在介导Nutlin-3这种生物学活性中的作用，其中针对p53的siRNA显著抵消了Nutlin-3的抗破骨细胞活性。Nutlin-3在SaOS-2骨肉瘤和破骨细胞前体细胞的共培养系统中也显著减少破骨细胞的形成。
这些发现表明，Nutlin-3通过p53依赖途径消除破骨细胞前体细胞的增殖和分化，可能对那些以破骨细胞活性异常为特征的肿瘤性疾病具有治疗意义。
