Density dependent modulation of cell cycle protein expression in astrocytes

The proliferation of type-1 astrocytes is strongly inhibited by homotypic cell-contact. To examine the mechanisms mediating this inhibition of proliferation, the expression of cell cycle related proteins was compared between exponential growth-phase and contact-inhibited astrocytes. Expression of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 was upregulated 10-fold in confluent compared with growth-phase cultures. Density-induced expression of p27Kip1 was reversible. When confluent cultures of astrocytes expressing high levels of p27Kip1 were replated at low density, the expression of p27Kip1 decreased rapidly. In contrast to p27Kip1, the expression levels of the cell cycle protein, cyclin A was decreased ten-fold in confluent cultures compared with those in growth phase. In addition, the ratio of hyperphosphorylated to hypophosphorylated retinoblastoma protein (pRb) decreased concomitantly with the increase of p27Kip1 and the decrease of cyclin A levels. These results suggest that increased expression of p27Kip1 and decreased expression of cyclin A underlie the reduction in proliferation of contact inhibited astrocytes. High levels of mitogenic stimulation could transiently override contact-dependent inhibition of astrocyte proliferation. Addition of exogenous epidermal growth factor (EGF) resulted in elevated proliferation at high density and formation of multiple cell layers. Addition of EGF did not substantially alter levels of p27Kip1 or cyclin A, but did elevate the levels of cyclin D1. Such changes in cell cycle protein expression may contribute to elevated cell proliferation seen in reactive gliosis after injury to the adult CNS.