GSK-3 can be regulated either by Wnt signaling or by the PI3-kinase/Akt pathway. The role of GSK-3 in the Wnt pathway is well-characterized, with GSK-3 acting to control gene expression by phosphorylating β-catenin (1
). As a target of PI 3-kinase/Akt signaling, GSK-3 plays a key role in regulating cell metabolism, proliferation and survival. However, although its targets include a variety of transcription factors, the transcriptional program controlled by GSK-3 downstream of PI 3-kinase/Akt signaling has not been elucidated. In the present study, we have employed a systems level approach to understanding the GSK-3 transcriptional program. We began with a set of 31 immediate-early genes we had previously characterized by global expression profiling as being induced by PI 3-kinase signaling downstream of PDGF stimulation of quiescent cells (11
). GSK-3 is active in quiescent cells and is inhibited by Akt phosphorylation, thus we sought to identify GSK-3-regulated genes by determining whether any of the PDGF-inducible PI 3-kinase dependent genes could be induced simply by inhibiting GSK-3 in the absence of growth factor stimulation. Twelve of these 31 PI 3-kinase dependent genes (~40%) were inducible by the direct inhibition of GSK-3 with the small molecule inhibitor SB-216763 in the absence of growth factor. These results indicate that the activity of GSK-3 plays a major role in signaling the repression of genes in quiescent cells.
Using computational methods, we then examined the upstream sequences of the 12 genes induced by GSK-3 inhibition. Our analysis focused on prediction of transcription factor binding sites that were over-represented in the group of GSK-3-regulated genes and were conserved between mouse and human orthologs. This analysis predicted binding sites for CREB as the most significantly overrepresented and evolutionarily conserved sites amongst the upstream sequences of GSK-3-regulated genes. This prediction is consistent with previous studies showing that CREB is phosphorylated by GSK-3 (27
). Moreover, the activity of CREB is generally inhibited by GSK-3 (28
), consistent with the induction of CREB-regulated genes following GSK-3 inhibition.
The upstream regions of 11 of the 12 GSK-3-regulated genes contained predicted CREB binding sites, and 9 of the 12 contained predicted CREB sites that were conserved between mouse and human. CREB binding to the upstream regions of 6 of these 9 genes was demonstrated by ChIP, providing experimental confirmation of the computational predictions. These results confirm previous demonstrations of CREB binding sites upstream of NR4A1
), as well as identifying new CREB binding sites upstream of CYR61
, and NR4A3
Proximal TATA boxes have been reported to be frequently present in the promoter regions of CREB-regulated genes (19
). Overall, 6 of the 9 SB-216763 induced genes with conserved CREB sites contained TATA boxes in their promoter regions, as compared to only about 21% of human transcripts in RefSeq (see Supplementary Fig. 2C). Notably, conserved TATA boxes were found upstream of 5 of the 6 genes to which CREB binding was demonstrated by ChIP. The combined prediction of both a conserved CREB site and a TATA box was thus highly correlated with CREB binding to the upstream region of a gene.
The physiological activity of these CREB binding sites was demonstrated by treatment of cells with forskolin, which activates the cAMP pathway. Forskolin induced transcription of 5 of the 6 genes with CREB binding sites (FOSB, NR4A1, NR4A2, NR4A3, and RGS1). In addition, forskolin led to recruitment of the CREB coactivator CBP to all 5 of these genes, as well as to the 6th gene to which CREB binding had been demonstrated (CYR61). It is interesting that CREB bound to the CYR61 upstream region, and that CBP binding was increased in response to forskolin, even though CYR61 was not induced by forskolin. Although CYR61 appears to contain a functional CREB site, it thus appears that activation of CREB is not sufficient for CYR61 induction.
Inhibition of GSK-3 also induced recruitment of CBP to the upstream regions of FOSB
. GSK-3 phosphorylates CREB at serine 129, and increased binding of CBP to CREB has been previously observed in cells following inhibition of GSK-3 (31
). It is also possible that phosphorylation by GSK3 affects the association of CREB with other coactivators such as p300 or members of the TORC family (33
A direct role for CREB in induction of all 5 of the forskolin-inducible genes was further demonstrated by RNA interference experiments, in which knockdown of CREB substantially inhibited (60-90%) the induction of these genes by forskolin. Importantly, RNAi against CREB also significantly inhibited (>80%) the induction of 3 of these genes (NR4A1
) resulting from inhibition of GSK-3. It is likely that CREB also contributes to induction of the other three genes following inhibition by GSK-3, but that knockdown of CREB is not sufficient to block induction due to the action of additional transcription factors targeted by GSK-3. Such factors might include members of the AP1 and CEBP families. Binding sites for these factors were also over-represented amongst the GSK-3 inducible genes (see ) and members of the AP1 and CEBP families have also been reported to be phosphorylated by GSK-3 (10
Taken together, these results indicate that CREB is a major target of PI 3-kinase/Akt/GSK-3 signaling during gene induction in response to growth factor stimulation. Previous studies have shown that regulation of CREB by GSK-3 plays a significant role in the Toll-like receptor inflammatory response downstream of PI 3-kinase in monocytes and macrophages (31
). Our results extend the role of GSK-3/CREB signaling to the more general paradigm of gene regulation downstream of growth factor receptors. GSK-3 is active under conditions of growth factor deprivation, which induce cell cycle arrest and/or apoptosis. In quiescent cells deprived of growth factors, it appears that GSK-3 actively represses gene expression, with inhibition of CREB playing a key role in this transcriptional response.