Reconstitution of Cyclin D1-Associated Kinase Activity Drives Terminally Differentiated Cells into the Cell Cycle

Abstract

Terminal cell differentiation entails definitive withdrawal from the cell cycle. Although most of the cells of an adult mammal are terminally differentiated, the molecular mechanisms preserving the postmitotic state are insufficiently understood. Terminally differentiated skeletal muscle cells, or myotubes, are a prototypic terminally differentiated system. We previously identified a mid-G₁ block preventing myotubes from progressing beyond this point in the cell cycle. In this work, we set out to define the molecular basis of such a block. It is shown here that overexpression of highly active cyclin E and cdk2 in myotubes induces phosphorylation of pRb but cannot reactivate DNA synthesis, underscoring the tightness of cell cycle control in postmitotic cells. In contrast, forced expression of cyclin D1 and wild-type or dominant-negative cdk4 in myotubes restores physiological levels of cdk4 kinase activity, allowing progression through the cell cycle. Such reactivation occurs in myotubes derived from primary, as well as established, C2C12 myoblasts and is accompanied by impairment of muscle-specific gene expression. Other terminally differentiated systems as diverse as adipocytes and nerve cells are similarly reactivated. Thus, the present results indicate that the suppression of cyclin D1-associated kinase activity is of crucial importance for the maintenance of the postmitotic state in widely divergent terminally differentiated cell types.

ACKNOWLEDGMENTS

We are grateful to J. Cook and J. Nevins for generously donating unpublished viruses. Our thanks go to T.-C. He and B. Vogelstein for the adenovirus construction system. We also thank S. van den Heuvel, G. Cossu, C. Schneider, G. Draetta, and J. Pines, who donated reagents that allowed us to perform the present work. We thank F. Tató for critically reading the manuscript.

A.S., D.P., and A.F. are recipients of FIRC fellowships. This work was supported by the Comitato Telethon Fondazione Onlus, the Associazione Italiana per la Ricerca sul Cancro, and the Italian Ministry of Health.

The first two authors contributed equally to this work.