Glycogen synthase kinase-3 is involved in the regulation of the cell cycle in cerebellar granule cells

Recent studies have demonstrated that neuronal reentry in the cell cycle and specifically the expression of the transcription factor E2F-1, constitutes a pathway that may be involved in neuronal apoptosis after serum and potassium withdrawal. Other enzymes such as glycogen synthase kinase-3 beta (GSK-3 beta) are also involved in this apoptotic stimulus, and thus in the process of neuronal cell death. Primary cerebellar granule cells (CGNs) were used in this study to determine whether pharmacological inhibition of GSK-3 beta is involved in neuronal modulation of the cell cycle, and specifically in the regulation of E2F-1 and retinoblastoma protein (Rb). CGNs showed a dramatic increase in GSK-3 beta activity after 2 h of serum and potassium deprivation. Immunoblot and activity assays revealed that lithium and SB415286 inhibit fully the activation of GSK-3 beta and attenuate the expression of cyclin D, cyclin E, pRb phosphorylation and the transcription factor E2F-1. These data were confirmed using AR-014418, a selective GSK-3 beta inhibitor that prevents the expression of cell-cycle proteins. Our data indicate that GSK-3 beta inhibition regulates, in part, the cell cycle in CGNs by inhibiting Rb phosphorylation and thus inhibiting E2F-1 activity. However, the selective inhibition of GSK-3 beta with AR-A014418 had not effect on cell viability or apoptosis mediated by S/K withdrawal. Furthermore, our results suggest that selective GSK-3 beta inhibition is not sufficient to protect against apoptosis in this S/K withdrawal model, indicating that Li+ and SB415286 neuroprotective effects are mediated by the inhibition of additional targets to GSK-3 beta. Therefore, there is a connection between cell cycle and GSK-3 beta activation and that these, along with other mechanisms, are involved in the molecular paths leading to the apoptotic process of rat CGNs triggered by S/K withdrawal. (C) 2007 Elsevier Ltd. All rights reserved.