A GSK-3-mediated transcriptional network maintains repression of immediate early genes in quiescent cells.

Glycogen synthase kinase-3 (GSK-3) plays a central role in cell survival and proliferation, in part by the regulation of transcription. Unlike most protein kinases, GSK-3 is active in quiescent cells in the absence of growth factor signaling. In a recent series of studies, we employed a systems-level approach to understanding the transcription network regulated by GSK-3 in a quiescent cell model. We identified a group of immediate early genes that were upregulated in quiescent cells solely by the inhibition of GSK-3 in the absence of growth factor stimulation. Computational analysis of the upstream sequences of these genes identified statistically over-represented binding sites for the transcription factors CREB, NFκB and AP-1, and the roles of these factors in regulating expression of GSK-3 target genes were verified by chromatin immunoprecipitation and RNA interference. In quiescent cells, GSK-3 inhibits CREB, NFκB and AP-1, thereby maintaining repression of their target genes and contributing to maintenance of cell cycle arrest.

AP-1 is a component of the transcriptional network regulated by GSK-3 in quiescent cells.

BACKGROUND: The protein kinase GSK-3 is constitutively active in quiescent cells in the absence of growth factor signaling. Previously, we identified a set of genes that required GSK-3 to maintain their repression during quiescence. Computational analysis of the upstream sequences of these genes predicted transcription factor binding sites for CREB, NFκB and AP-1. In our previous work, contributions of CREB and NFκB were examined. In the current study, the AP-1 component of the signaling network in quiescent cells was explored. METHODOLOGY/PRINCIPAL FINDINGS: Using chromatin immunoprecipitation analysis, two AP-1 family members, c-Jun and JunD, bound to predicted upstream regulatory sequences in 8 of the 12 GSK-3-regulated genes. c-Jun was phosphorylated on threonine 239 by GSK-3 in quiescent cells, consistent with previous studies demonstrating inhibition of c-Jun by GSK-3. Inhibition of GSK-3 attenuated this phosphorylation, resulting in the stabilization of c-Jun. The association of c-Jun with its target sequences was increased by growth factor stimulation as well as by direct GSK-3 inhibition. The physiological role for c-Jun was also confirmed by siRNA inhibition of gene induction. CONCLUSIONS/SIGNIFICANCE: These results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-inducible genes in quiescent cells. Together, AP-1, CREB and NFκB form an integrated transcriptional network that is largely responsible for maintaining repression of target genes downstream of GSK-3 signaling.

GSK-3 represses growth factor-inducible genes by inhibiting NF-kappaB in quiescent cells.

GSK-3 is active in the absence of growth factor stimulation and generally acts to induce apoptosis or inhibit cell proliferation. We previously identified a subset of growth factor-inducible genes that can also be induced in quiescent T98G cells solely by inhibition of GSK-3 in the absence of growth factor stimulation. Computational predictions verified by chromatin immunoprecipitation assays identified NF-kappaB binding sites in the upstream regions of 75% of the genes regulated by GSK-3. p50 bound to most of these sites in quiescent cells, and for one-third of the genes, binding of p65 to the predicted sites increased upon inhibition of GSK-3. The functional role of p65 in gene induction following inhibition of GSK-3 was demonstrated by RNA interference experiments. Furthermore, inhibition of GSK-3 in quiescent cells resulted in activation of IkappaB kinase, leading to phosphorylation and degradation of IkappaB alpha and nuclear translocation of p65 and p50. Taken together, these results indicate that the high levels of GSK-3 activity in quiescent cells repress gene expression by negatively regulating NF-kappaB through inhibition of IkappaB kinase. This inhibition of NF-kappaB is consistent with the role of GSK-3 in the induction of apoptosis or cell cycle arrest in cells deprived of growth factors.

Phosphatidylinositol 3-kinase signaling in proliferating cells maintains an anti-apoptotic transcriptional program mediated by inhibition of FOXO and non-canonical activation of NFkappaB transcription factors.

BACKGROUND: Phosphatidylinositol (PI) 3-kinase is activated by a variety of growth factor receptors and the PI 3-kinase/Akt signaling pathway is a key regulator of cell proliferation and survival. The downstream targets of PI 3-kinase/Akt signaling include direct regulators of cell cycle progression and apoptosis as well as a number of transcription factors. Growth factor stimulation of quiescent cells leads to robust activation of PI 3-kinase, induction of immediate-early genes, and re-entry into the cell cycle. A lower level of PI 3-kinase signaling is also required for the proliferation and survival of cells maintained in the presence of growth factors, but the gene expression program controlled by PI 3-kinase signaling in proliferating cells has not been elucidated. RESULTS: We used microarray analyses to characterize the changes in gene expression resulting from inhibition of PI 3-kinase in proliferating cells. The genes regulated by inhibition of PI 3-kinase in proliferating cells were distinct from genes induced by growth factor stimulation of quiescent cells and highly enriched in genes that regulate programmed cell death. Computational analyses followed by chromatin immunoprecipitations demonstrated FOXO binding to both previously known and novel sites in promoter regions of approximately one-third of the up-regulated genes, consistent with activation of FOXO1 and FOXO3a in response to inhibition of PI 3-kinase. NFkappaB binding sites were similarly identified in promoter regions of over one-third of the down-regulated genes. RelB was constitutively bound to promoter regions in cells maintained in serum, however binding decreased following PI 3-kinase inhibition, indicating that PI 3-kinase signaling activates NFkappaB via the non-canonical pathway in proliferating cells. Approximately 70% of the genes targeted by FOXO and NFkappaB regulate cell proliferation and apoptosis, including several regulators of apoptosis that were not previously known to be targeted by these transcription factors. CONCLUSION: PI 3-kinase signaling in proliferating cells regulates a novel transcriptional program that is highly enriched in genes that regulate apoptosis. At least one-third of these genes are regulated either by FOXO transcription factors, which are activated following PI 3-kinase inhibition, or by RelB, which is activated by PI 3-kinase via the non-canonical pathway in proliferating cells.

Immediate-early and delayed primary response genes are distinct in function and genomic architecture.

The transcriptional program induced by growth factor stimulation is classically described in two stages as follows: the rapid protein synthesis-independent induction of immediate-early genes, followed by the subsequent protein synthesis-dependent induction of secondary response genes. In this study, we obtained a comprehensive view of this transcriptional program. As expected, we identified both rapid and delayed gene inductions. Surprisingly, however, a large fraction of genes induced with delayed kinetics did not require protein synthesis and therefore represented delayed primary rather than secondary response genes. Of 133 genes induced within 4 h of growth factor stimulation, 49 (37%) were immediate-early genes, 58 (44%) were delayed primary response genes, and 26 (19%) were secondary response genes. Comparison of immediate-early and delayed primary response genes revealed functional and regulatory differences. Whereas many immediate-early genes encoded transcription factors, transcriptional regulators were not prevalent among the delayed primary response genes. The lag in induction of delayed primary response compared with immediate-early mRNAs was because of delays in both transcription initiation and subsequent stages of elongation and processing. Consistent with increased abundance of RNA polymerase II at their promoters, immediate-early genes were characterized by over-representation of transcription factor binding sites and high affinity TATA boxes. Immediate-early genes also had short primary transcripts with few exons, whereas delayed primary response genes more closely resembled other genes in the genome. These findings suggest that genomic features of immediate-early genes, in contrast to the delayed primary response genes, are selected for rapid induction, consistent with their regulatory functions.

Glycogen synthase kinase-3 represses cyclic AMP response element-binding protein (CREB)-targeted immediate early genes in quiescent cells.

Despite its central role in cell survival and proliferation, the transcriptional program controlled by GSK-3 is poorly understood. We have employed a systems level approach to characterize gene regulation downstream of PI 3-kinase/Akt/GSK-3 signaling in response to growth factor stimulation of quiescent cells. Of 31 immediate-early genes whose induction was dependent on PI 3-kinase signaling, 12 were induced directly by inhibition of GSK-3. Most of the GSK-3-regulated genes encoded transcription factors, growth factors, and signaling molecules. Binding sites for CREB were highly over-represented in the upstream regions of these genes, with 9 genes containing CREB sites that were conserved in mouse orthologs. Binding sites predicted in 6 genes were confirmed by CREB chromatin immunoprecipitation and forskolin induction of CBP binding. Moreover, CREB siRNA substantially blocked induction of 5 genes by forskolin and of 3 genes following inhibition of GSK-3. These results indicate that GSK-3 actively represses gene expression in quiescent cells, with inhibition of CREB playing a key role in this transcriptional response.

Identification of transcription factor binding sites upstream of human genes regulated by the phosphatidylinositol 3-kinase and MEK/ERK signaling pathways.

We have taken an integrated approach in which expression profiling has been combined with the use of small molecule inhibitors and computational analysis of transcription factor binding sites to characterize regulatory sequences of genes that are targets of specific signaling pathways in growth factor-stimulated human cells. T98G cells were stimulated with platelet-derived growth factor (PDGF) and analyzed by DNA microarrays, which identified 74 immediate-early gene transcripts. Cells were then treated with inhibitors to identify subsets of genes that are targets of the phosphatidylinositol 3-kinase (PI3K) and MEK/ERK signaling pathways. Four groups of PDGF-induced genes were defined: independent of PI3K and MEK/ERK signaling, dependent on PI3K signaling, dependent on MEK/ERK signaling, and dependent on both pathways. The upstream regions of all genes in the four groups were scanned using TRANSFAC for putative cis-elements as compared with a background set of non-induced genes. Binding sites for 18 computationally predicted transcription factors were over-represented in the four groups of co-expressed genes compared with the background sequences (p < 0.01). Many of the cis-elements identified were conserved in orthologous mouse genes, and many of the predicted elements and their cognate transcription factors were consistent with previous experimental data. In addition, chromatin immunoprecipitation assays experimentally verified nine predicted SRF binding sites in T98G cells, including a previously unknown SRF site upstream of DUSP5. These results indicate that groups of human genes regulated by discrete intracellular signaling pathways share common cis-regulatory elements.