Objectives: 

Cell cycle inhibitors are known to be implicated in several biological processes beyond cycle regulation. In fact, some of them have been widely described to play roles in the differentiation of a number of cell types as well as in cell fate determination. Specifically, members of the pocket protein family, p107 and p130, along with cyclin-dependent kinase inhibitor p27, participate in the control of chondrocyte hypertrophic differentiation. Moreover, both p130 and p27 seem to have overlapping activities with p107 in the context of chondrogenic proliferation and maturation.

Materials: 

Upon treatment with insulin, chondrogenic ATDC5 cell line undergoes a multistep process of differentiation that mimics the process occurred during endochondral bone formation. Thus, we employed this cell system to study the contribution of cell cycle regulators to this process. In this work, we used lentiviral-mediated shRNA delivery to create ATDC5 cells pools lacking p107, p130 and p27, alone or in combination. Populations of different genotypes were then characterised in terms of proliferation properties as well as differentiation potential.

Results: 

Our data show that the absence of p107, p130 or p27 causes an increase in proliferation, and a defective cell growth arrest after treatment with differentiation signals. Knockdown of p107 had a greater impact than the depletion of p130 or p27 alone. On the other hand, the process of chondrogenic maturation is affected in ATDC5 model by the absence of p107, p130 or p27 in a similar extent, as those cells fail to generate ECM at normal rates. In agreement with previous results in animal models, combined loss of p107 and p130 caused deeper defects than the lack of p107 or 130 alone.

Conclusions: 

Taken together, the results obtained in our cell systems are consistent with previous observations in genetically-engineered mouse models. Populations of ATDC5 carrying combinations of knockdowns constitute a good model for the in vitro dissection of cell cycle inhibitors contribute to endochondral bone formation.