The PI 3-kinase/Akt/GSK-3 signaling pathway plays a central role in regulation of growth factor-dependent proliferation and survival of mammalian cells, at least in part by transcriptional regulation. A number of transcription factors and target genes that are regulated by PI 3-kinase signaling have been studied on an individual basis. In addition, both we [26
] and others [27
] have used global expression profiling to investigate the overall program of gene regulation controlled by PI 3-kinase/Akt/GSK-3 signaling. These studies have focused on the response of quiescent cells to growth factor stimulation, which induces robust activation of PI 3-kinase, induction of immediate-early genes, and re-entry into the cell cycle. However, a lower level of continuous PI 3-kinase signaling is also required to maintain proliferation and survival of cells in the presence of serum growth factors. In the present study, we have therefore undertaken a global analysis of the transcriptional program that maintains cell survival and proliferation downstream of PI 3-kinase signaling in proliferating cells.
Inhibition of PI 3-kinase in actively proliferating T98G cells for 2 and 4 hours resulted in up-regulation of 32 genes and down-regulation of 53 genes. These gene expression changes occurred around the time at which apoptosis was first detected. Consistent with their involvement in apoptosis, the genes with altered expression following 2 and 4 hours of PI 3-kinase inhibition included growth factors and cytokines, as well as several well-known regulators of apoptosis (such as DDIT3/CHOP, GADD45B, PHLDA1, BIRC3/cIAP2 and TNFRSF11B/osteoprotegerin) and cell cycle progression (such as CCNG2 and CCND1). Functional classifications using the Gene Ontology database further indicated that these gene sets were enriched in terms related to cell stress and programmed cell death, as well as to cell signaling and transcriptional regulation. The numbers of up- and down-regulated genes both increased substantially, to a total of nearly 250 genes, after 8 hours of PI 3-kinase inhibition, probably reflecting a secondary transcriptional response. This larger set of genes was no longer significantly enriched in functional GO terms.
We compared the sets of genes with altered expression following 2 and 4 hours of PI 3-kinase inhibition to those identified by previous studies of the genes induced following 0.5–4 hours of growth factor simulation of quiescent T98G cells [26
]. Only ~10% of the genes affected by inhibition of PI 3-kinase overlapped with either the total sets of immediate-early or delayed primary response genes, or with the subset of PI 3-kinase dependent immediate-early genes defined in these previous studies. Similar to the genes that were up-regulated following inhibition of PI 3-kinase, immediate-early genes induced in response to growth factor stimulation were enriched in GO terms related to transcriptional regulation [44
]. However, the transcription factors induced as immediate-early genes by growth factor stimulation are different from those that are up-regulated in response to PI 3-kinase inhibition. The genes regulated by continuous PI 3-kinase signaling in proliferating cells were thus clearly distinct from the primary response genes induced by growth factor stimulation of quiescent cells.
We also compared the genes affected by PI 3-kinase inhibition with the genes identified in a recent study of the effects of mitogen withdrawal on gene expression in proliferating human fibroblasts [65
]. In these experiments, primary fetal human lung fibroblasts were cultured in the presence of a low concentration of serum, IGF-1 and PDGF, and were growth-arrested by withdrawal of PDGF, which resulted in the entry of cells into G0
; however, in contrast to our experiments, no significant increase in cells undergoing apoptosis was observed. This entry of cells into quiescence rather than apoptosis is consistent with the continued presence of low concentrations of serum and IGF-1 in the experiments of Coller et al
], in contrast to the complete inhibition of PI 3-kinase in our studies. Comparing these two studies indicated that very little overlap exists between the genes regulated by inhibition of PI 3-kinase signaling and those regulated by 14 hours or 4 days of PDGF withdrawal. However, most of the overlapping genes, including CCNG2, PLAU, BDNF
, and FGF2
, and a number of non-overlapping genes regulated by mitogen withdrawal have been connected to the regulation of cell proliferation. Thus, there are functional overlaps in the genes regulated by PI 3-kinase inhibition and mitogen withdrawal, although they are clearly distinct gene sets as might be expected from the differences in treatments and their distinct effects on proliferation and apoptosis. We note that asynchronous cells were used in our study, and that similar analyses of synchronized cells might reveal PI 3-kinase regulation of distinct subsets or additional genes during specific stages of the cell cycle.
We have previously used computational analysis to identify transcription factor binding sites that are over-represented in promoter regions of immediate-early genes, including those regulated by PI 3-kinase signaling [26
]. The transcription factors and families involved in regulation of these genes included CREB, SRF, FOXO and NFκB. We applied a similar analysis to the gene sets affected by inhibition of PI 3-kinase signaling in the present study. This revealed over-representation of phylogenetically conserved binding sites for FOXO and NFκB upstream of the genes that were up- and down-regulated, respectively, following 2 and 4 hours of PI 3-kinase inhibition. Many of the predicted FOXO and NFκB binding sites were confirmed by ChIP, indicating that the FOXO and NFκB transcription factors play major roles in the transcriptional response to PI 3-kinase inhibition. This is consistent with the established regulation of these transcription factors by PI 3-kinase/Akt signaling, which inhibits FOXO and promotes NFκB activity [16
]. Since p53 is also a major target of PI 3-kinase/Akt signaling in control of cell proliferation and survival [12
], we might have expected to similarly observe an over-representation of p53 binding sites. However, p53
is inactivated by mutation in T98G cells [66
], accounting for its absence in the gene sets analyzed in our experiments.
In total, we identified FOXO and NFκB binding sites upstream of about one-third of the genes that were up- and down-regulated, respectively, by inhibition of PI 3-kinase. Since our computational predictions of transcription factor binding sites were limited to phylogenetically conserved sites in promoter regions, this may be an underestimate of the total fraction of PI 3-kinase regulated genes that are targeted by these transcription factors. In particular, additional genes may be regulated by FOXO or NFκB binding sites in enhancers rather than promoter sequences, or by sites that were not conserved in the mouse. Other genes may be regulated at the transcriptional level by other transcription factors (such as Myc/Max) or by non-transcriptional mechanisms, including regulation of mRNA decay [27
Two members of the FOXO family (FOXO1 and 3a) were expressed in T98G cells and, as expected, both were activated in response to PI 3-kinase inhibition. In contrast, there are 5 members of the NFκB family (p65, c-Rel, RelB, p50 and p52), which can be activated by either the canonical or non-canonical pathways, both of which are stimulated by Akt [19
]). We initially demonstrated binding of p65 to predicted NFκB sites following stimulation of the canonical pathway by TNFα. In unstimulated cells growing in the presence of serum however, only binding of p50, p52 and RelB was detected by ChIP, indicating that just the non-canonical pathway was active. Of these three family members, RelB is the main effector of this pathway, as it is the only one that contains a transactivation domain. Since the binding of RelB to promoters of down-regulated genes decreased following inhibition of PI 3-kinase, it appears that PI 3-kinase signaling regulates the non-canonical pathway of NFκB activation in proliferating cells.
The 10 genes identified as targets of FOXO and the 18 identified as targets of NFκB are summarized in Table . Binding sites for FOXO had been previously identified upstream of 4 of these genes, and new sites were identified upstream of 6 genes in the present study. Similarly, the 22 NFκB binding sites identified upstream of 18 genes included 10 that were previously known, 5 that were newly identified in genes previously known to be regulated by NFκB, and 7 upstream of new NFκB target genes identified in the present study. It is noteworthy that nearly half of the FOXO and NFκB target genes whose expression is altered in response to inhibition of PI 3-kinase are known to function as regulators of apoptosis, including the previously unrecognized FOXO targets DDIT3/CHOP and TXNIP and the NFκB targets AMIGO2, BDNF, IGFBP5, and TNFRSF11B/osteoprotegerin. The predominant functions of these PI 3-kinase regulated genes emphasize the critical role of PI 3-kinase/Akt regulation of the FOXO and NFκB transcription factors in maintaining cell survival.
FOXO and NFκB regulated genes.