Expression of Cell Cycle Regulatory Factors in Differentiating Osteoblasts: Postproliferative Up-Regulation of Cyclins B and E1

The representation of cyclins and cyclin-dependent kinases (cdks) was analyzed during progressive development of the bone cell phenotype in cultures of normal diploid rat calvarÃ-a!osteoblasts. Three developmental stages were examined: (a) proliferation; (In monolayer confluency; and ic i mineralization of the bone extracellular matrix. We demonstrate that the presence of cyclins and cdks is not restricted to the proliferation period. Consistent with their role in cell cycle progression, cdc2 and cdk2 decrease postproliferatively. However, cdk4 and cyclins A, B, and Dl persist in confluent cells. Cyclin E is significantly up-regulated during the extracellular matrix mineralization developmental period. Examination of the cytoplasmic levels of these cell cycle regulatory proteins indicates a marked increase in cyclin B in the late differentiation stage. The elevation of nuclear cyclin E and cytoplasmic cyclin B is not observed in osteoblasts maintained under culture conditions that do not support differentiation. Furthermore, treatment with transforming growth factor /! for 48 h during the proliferation period renders the cells incompetent for differ entiation and abrogates the postproliferative up-regulation of cyclins B and E. Density-induced growth inhibition of KOS 17/2.8 osteosarcoma cells is not accompanied by up-regulation of nuclear cyclin E and cyto plasmic cyclin B when compared to the proliferation period. This obser vation is consistent with abrogation of both growth control and differen tiation regulatory mechanisms in tumor cells. These results suggest that cell cycle regulatory proteins function not only during proliferation but may also play a role in normal diploid osteoblast differentiation.