p65 Apoptosis p105 NF-κB Cytochrome c release TNF signaling cascade Caspase cascade activation Figure 3: GSK-3β’s role in apoptosis signaling. The above schematic shows the role of activated GSK-3β and its role in regulating apoptosis. Active GSK-3β inhibits MDM2 regulation of p53, leading to DNA repair and growth arrest, and in some cases the activation of the caspase cascade through Bax to promote apoptosis. Active GSK-3β also positively regulates NFκB by activating IKK, IκB, and p65, leading to the inhibition of TNF-mediated apoptosis. These actions inhibit the initiation of apoptosis through the TNF signaling cascade. Similar protection from GSK-3β inhibition has been seen in primary neurons [38]. The mechanism of protection from radiation-induced apoptosis in these cells involves subcellular localization and interaction of GSK-3β, p53, and MDM2. GSK-3β inhibition blocks radiation-induced accumulation of p53 by upregulating levels of MDM2 that subsequently result in decreased radiation-dependent apoptosis [71]. In addition to abrogation of radiation- induced p53 phosphorylation, accumulation, and nuclear translocation, GSK-3β inhibition results in the accumulation of MDM2 and sequestration of GSK-3β, p53, and MDM2 in the cytoplasm where p53 cannot act on its target genes [71]. The role of attenuated p53 function in the prosurvival eﬀects of the GSK-3β inhibitors, has also been previously described [34, 46, 70, 72, 73]. In regulation of the apoptotic response, mammalian cells employ multiple prosurvival proteins from the Bcl-2 family (Bcl-2, Bcl-XL, Bcl-w, Mcl1, and A1) that antagonize the proapoptotic function of Bax and Bak [34, 74]. Bax and Bak localize to the mitochondrial outer membrane and trigger death signals leading to cytochrome c release to the cytosol [74, 75]. Apoptosis requires a group of eﬀector caspases to dismantle the cells. Cytochrome c activates caspase-9, which subsequently activates caspase-3 [76]. The activation of caspase-3 is an essential step leading to cleavage of the DNA repair enzyme, poly (ADP-ribose) polymerase (PARP), resulting in genomic DNA fragmentation. Bax protein levels and cleavage (activation) of caspase-3 were increased due to radiation and were abrogated by GSK-3β inhibitors [77] (Figure 3). GSK-3β was also found to be associated with 4 Growth arrest DNA repair MDM2 p53 Bax GSK-3β International Journal of Cell Biology mitochondrial apoptotic signaling. Inhibition of GSK-3β prevented mitochondrial release of cytochrome c, which is known to activate caspase-3 and initiate apoptosis [34]. Phosphatidylinositol 3-kinase (PI3-kinase) and its down- stream eﬀector, the protein-serine/threonine kinase Akt, a negative regulator of GSK-3β, play an important role in preventing apoptosis by blocking activation of the caspase cascade [78]. 3. Survival-Promoting Effects of GSK-3β GSK-3β is involved in multiple signaling pathways and has many phosphorylation targets. It should therefore not be surprising that GSK-3β has both pro- and antiapoptotic roles. The overall eﬀect of GSK-3β on cell survival varies depending on cell type, transformation status, and the speciﬁc signaling pathway being activated. For example, despite evidence for a substantial proapoptotic role of GSK- 3β, it is the inhibition of GSK-3β that promotes apoptosis and decreases viability in neuroblastoma cells [79]. Several examples of pro-survival roles of GSK-3β not mentioned here are summarized in Table 2 [80–84]. Additionally, while GSK-3β has been typically identiﬁed as an activator of p53-mediated apoptosis [34], conﬂicting reports suggest an inhibitory eﬀect of GSK-3β signaling on p53 activation. Inhibition of GSK-3β blocks activation of MDM2 by reducing Ser-254 phosphorylation. This prevents p53 degradation and promotes apoptosis despite the induc- tion of p53 ubiquitination. Similarly, ionizing radiation was found to induce an inactivating phosphorylation at Ser-9 of GSK-3β, corresponding to hypophosphorylation of MDM2 and accumulation of p53 [69]. In contrast to its proapoptotic eﬀects, this data suggests that GSK-3β inhibits apoptosis under basal conditions through MDM2-dependent degra- dation of p53. Overexpression of β-catenin, a downstream signaling factor negatively regulated by GSK-3β, was found to increase basal p53 levels by blocking both MDM2- dependent and independent degradation in neuroblastoma cells [85], providing additional supporting evidence for an inhibitory eﬀect of GSK-3β on p53-mediated apoptosis. Interestingly, a negative feedback loop exists between β- catenin and p53; while β-catenin upregulates p53 levels, the activation of p53 results in degradation of β-catenin through GSK-3β [86]. While the majority of publications suggest a proapoptotic role for GSK-3β in p53 signaling, it is clear that more comprehensive studies are needed in order to fully understand the p53-GSK-3β relationship. GSK-3β is speciﬁcally required for hepatocyte survival in normal embryos, and GSK-3β knockout mice are embry- onically lethal between E13.15–14.5. Hepatocyte apoptosis in GSK-3β knockout mice and mouse embryonic ﬁbrob- lasts results only after exposure to tumor necrosis factor (TNF), while inhibition of GSK-3β in wild-type cells with lithium increases TNF sensitivity. GSK-3β loss in these cells has a detrimental eﬀect on the action of NF-κB, which protects against TNF-induced apoptosis [88]. Other studies have shown that GSK-3β directly promotes NF-κB stability and activation through both the degradation of p105 and