Differential modulation of Akt/glycogen synthase kinase-3beta pathway regulates apoptotic and cytoprotective signaling responses.

We have previously reported that specific dopamine agonists mediate protection against apoptosis induced by oxidative stress by activating the D2 receptor-coupled phosphoinositide 3-kinase (PI-3K)/Akt pathway. In the present study we examined the downstream effectors of PI-3K/Akt signaling and their role in cell death after oxidative stress and protection provided by ropinirole, a D2 receptor agonist in PC12 cells and primary cultures of dopamine neurons. Ropinirole treatment was associated with rapid translocation and phosphorylation of the PI-3K substrate Akt and phosphorylation of Akt substrates. One of these Akt downstream substrates was identified as the pro-apoptotic factor glycogen synthase kinase-3beta (GSK-3beta). Ropinirole-induced protection was associated with phosphorylation of GSK-3beta (inactivation). In contrast, inhibition of PI-3K blocked the phosphorylation of Akt and GSK-3beta (activation) and prevented the protection mediated by ropinirole. Suppression of Akt with specific short hairpin RNA in normal PC12 cells caused cell death, which was associated with reduced phosphorylation of GSK-3beta and reduced levels of beta-catenin, a transcriptional activator that is regulated by GSK-3beta. Knock-out of GSK-3beta expression with a short hairpin RNA alone was itself sufficient to cause cell death. We further demonstrated that oxidative stress induced by hydrogen peroxide (H2O2) dephosphorylates Akt and GSK-3beta, increases GSK-3beta activity, and promotes an interaction with beta-catenin and its degradation. Inhibition of GSK-3beta activity by inhibitor VIII protects cells from H2O2 similar to ropinirole. These results indicate that GSK-3beta downstream of Akt plays a critical role in cell death and survival in these models.