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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.

Terragni J, Graham JR, Adams KW, Schaffer ME, Tullai JW, Cooper GM - BMC Cell Biol. (2008)

Bottom Line: We used microarray analyses to characterize the changes in gene expression resulting from inhibition of PI 3-kinase in proliferating cells.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Boston University, Boston MA 02215, USA. jolyon@bu.edu

ABSTRACT

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.

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PI 3-kinase inhibition causes a decrease in RelB binding. T98G cells were either maintained in serum or treated with 50 μM LY294002 for 4 or 8 hours. Chromatin fragments were immunoprecipitated with anti-RelB antibody and quantified by real-time PCR. Data are presented as the percentage of input and are the means of 3 independent experiments ± S.E. β-globin was used as the negative control. (*) represents statistically significant loss of binding compared to the cells maintained in serum (assessed by t test).
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Figure 7: PI 3-kinase inhibition causes a decrease in RelB binding. T98G cells were either maintained in serum or treated with 50 μM LY294002 for 4 or 8 hours. Chromatin fragments were immunoprecipitated with anti-RelB antibody and quantified by real-time PCR. Data are presented as the percentage of input and are the means of 3 independent experiments ± S.E. β-globin was used as the negative control. (*) represents statistically significant loss of binding compared to the cells maintained in serum (assessed by t test).

Mentions: Because RelB has a transactivation domain and the predicted sites occurred upstream of genes that were down-regulated in response to PI 3-kinase inhibition, we next sought to determine if RelB binding decreased in cells that were treated with LY294002 to inhibit PI 3-kinase. ChIP assays were performed in triplicate to measure RelB binding to the confirmed binding sites in the 7 genes mentioned previously (Fig. 7). Inhibition of PI 3-kinase led to a significant decrease in RelB binding (p < 0.05) to the sites in BIRC3, CCL2 and PTX3, consistent with PI 3-kinase regulation of these genes through the non-canonical NFκB pathway.


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.

Terragni J, Graham JR, Adams KW, Schaffer ME, Tullai JW, Cooper GM - BMC Cell Biol. (2008)

PI 3-kinase inhibition causes a decrease in RelB binding. T98G cells were either maintained in serum or treated with 50 μM LY294002 for 4 or 8 hours. Chromatin fragments were immunoprecipitated with anti-RelB antibody and quantified by real-time PCR. Data are presented as the percentage of input and are the means of 3 independent experiments ± S.E. β-globin was used as the negative control. (*) represents statistically significant loss of binding compared to the cells maintained in serum (assessed by t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2268685&req=5

Figure 7: PI 3-kinase inhibition causes a decrease in RelB binding. T98G cells were either maintained in serum or treated with 50 μM LY294002 for 4 or 8 hours. Chromatin fragments were immunoprecipitated with anti-RelB antibody and quantified by real-time PCR. Data are presented as the percentage of input and are the means of 3 independent experiments ± S.E. β-globin was used as the negative control. (*) represents statistically significant loss of binding compared to the cells maintained in serum (assessed by t test).
Mentions: Because RelB has a transactivation domain and the predicted sites occurred upstream of genes that were down-regulated in response to PI 3-kinase inhibition, we next sought to determine if RelB binding decreased in cells that were treated with LY294002 to inhibit PI 3-kinase. ChIP assays were performed in triplicate to measure RelB binding to the confirmed binding sites in the 7 genes mentioned previously (Fig. 7). Inhibition of PI 3-kinase led to a significant decrease in RelB binding (p < 0.05) to the sites in BIRC3, CCL2 and PTX3, consistent with PI 3-kinase regulation of these genes through the non-canonical NFκB pathway.

Bottom Line: We used microarray analyses to characterize the changes in gene expression resulting from inhibition of PI 3-kinase in proliferating cells.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Boston University, Boston MA 02215, USA. jolyon@bu.edu

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus