MDM2 antagonist Nutlin-3 suppresses the proliferation and differentiation of human pre-osteoclasts through a p53-dependent pathway.
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.


BACKGROUND
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.


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.
Zauli
Giorgio
G
Department of Biomedicine, University of Trieste, Trieste, Italy.
Rimondi
Erika
E
Corallini
Federica
F
Fadda
Roberto
R
Capitani
Silvano
S
Secchiero
Paola
P
D002448:Cell Adhesion / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D019070:Cell Lineage / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D012516:Osteosarcoma / Q000473:pathology; D010879:Piperazines / Q000494:pharmacology*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D053245:RANK Ligand / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects
D007093:Imidazoles; D010879:Piperazines; D053245:RANK Ligand; D016159:Tumor Suppressor Protein p53; C482205:nutlin 3; D016173:Macrophage Colony-Stimulating Factor; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2