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EGF-induced activation of Akt results in mTOR-dependent p70S6 kinase phosphorylation and inhibition of HC11 cell lactogenic differentiation.

Galbaugh T, Cerrito MG, Jose CC, Cutler ML - BMC Cell Biol. (2006)

Bottom Line: The activation of p70S6K by insulin or EGF resulted in the phosphorylation of ribosomal protein S6 (RPS6), elongation initiation factor 4E (elF4E) and 4E binding protein1 (4E-BP1).PI-3-kinase signaling contributes to the EGF block of lactogenic differentiation via Akt and p70S6K.The EGF-induced activation of PI-3-kinase-Akt-mTOR regulates phosphorylation of molecules including ribosomal protein S6, eIF4E and 4E-BP1 that influence translational control in HC11 cells undergoing lactogenic differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, United States Military Cancer Institute, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA. t-galbaugh@northwestern.edu

ABSTRACT

Background: HC11 mouse mammary epithelial cells differentiate in response to lactogenic hormone resulting in expression of milk proteins including beta-casein. Previous studies have shown that epidermal growth factor (EGF) blocks differentiation not only through activation of the Ras/Mek/Erk pathway but also implicated phosphatidylinositol-3-kinase (PI-3-kinase) signaling. The current study analyzes the mechanism of the PI-3-kinase pathway in an EGF-induced block of HC11 lactogenic differentiation.

Results: HC11 and HC11-luci cells, which contain luciferase gene under the control of a beta-casein promotor, were treated with specific chemical inhibitors of signal transduction pathways or transiently infected/transfected with vectors encoding dominant negative-Akt (DN-Akt) or conditionally active-Akt (CA-Akt). The expression of CA-Akt inhibited lactogenic differentiation of HC11 cells, and the infection with DN-Akt adenovirus enhanced beta-casein transcription and rescued beta-casein promotor-regulated luciferase activity in the presence of EGF. Treatment of cells with Rapamycin, an inhibitor of mTOR, blocked the effects of EGF on beta-casein promotor driven luciferase activity as effectively as PI-3-kinase inhibitors. While expression of CA-Akt caused a constitutive activation of p70S6 kinase (p70S6K) in HC11 cells, the inhibition of either PI-3-kinase or mTOR abolished the activation of p70S6K by EGF. The activation of p70S6K by insulin or EGF resulted in the phosphorylation of ribosomal protein S6 (RPS6), elongation initiation factor 4E (elF4E) and 4E binding protein1 (4E-BP1). But lower levels of PI-3-K and mTOR inhibitors were required to block insulin-induced phosphorylation of RPS6 than EGF-induced phosphorylation, and insulin-induced phosphorylation of elF4E and 4E-BP1 was not completely mTOR dependent suggesting some diversity of signaling for EGF and insulin. In HC11 cells undergoing lactogenic differentiation the phosphorylation of p70S6K completely diminished by 12 hours, and this was partly attributable to dexamethasone, a component of lactogenic hormone mix. However, p70S6K phosphorylation persisted in the presence of lactogenic hormone and EGF, but the activation could be blocked by a PI-3-kinase inhibitor.

Conclusion: PI-3-kinase signaling contributes to the EGF block of lactogenic differentiation via Akt and p70S6K. The EGF-induced activation of PI-3-kinase-Akt-mTOR regulates phosphorylation of molecules including ribosomal protein S6, eIF4E and 4E-BP1 that influence translational control in HC11 cells undergoing lactogenic differentiation.

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The effect of conditionally active-Akt1 and dominant negative-Akt1 on HC11 differentiation. A. The HC11-luci cells transiently transfected with either a conditionally active-Akt-1 (CA-Akt) or a control vector (pCDNA3.1). At 24 hours the cells were incubated in DIP-induction media with or without tamoxifen (1 μM). Luciferase activity was determined 48 hours post-induction and was normalized to protein concentration. Expression of Akt and HA-CA-Akt was determined via western blot. Lanes were loaded with equal amounts of protein (117.5 μg). B. The effect of dominant negative-Akt1 (DN-Akt) adenoviral infection on EGF disruption of differentiation. HC11-luci cells were infected with either a DN-Akt1 or control (LacZ) adenovirus. Cells were changed to DIP-induction media the next day and lysates were harvested 48 hours post-induction for β-casein promotor luciferase activity. The results were compared to cultures exposed to DIP plus LY294002 (5 μM). Expression of Akt was determined via western blot analysis and gels were loaded with equal amount of protein (120 μg). C: HC11 cells were infected with either a DN-Akt1 or control (LacZ) adenovirus. RNA was harvested 48 hours post-induction for analysis of β-casein RNA expression by northern blot. Expression of Akt was determined via western blot analysis and gels were loaded with equal amount of protein (120 μg).
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Figure 2: The effect of conditionally active-Akt1 and dominant negative-Akt1 on HC11 differentiation. A. The HC11-luci cells transiently transfected with either a conditionally active-Akt-1 (CA-Akt) or a control vector (pCDNA3.1). At 24 hours the cells were incubated in DIP-induction media with or without tamoxifen (1 μM). Luciferase activity was determined 48 hours post-induction and was normalized to protein concentration. Expression of Akt and HA-CA-Akt was determined via western blot. Lanes were loaded with equal amounts of protein (117.5 μg). B. The effect of dominant negative-Akt1 (DN-Akt) adenoviral infection on EGF disruption of differentiation. HC11-luci cells were infected with either a DN-Akt1 or control (LacZ) adenovirus. Cells were changed to DIP-induction media the next day and lysates were harvested 48 hours post-induction for β-casein promotor luciferase activity. The results were compared to cultures exposed to DIP plus LY294002 (5 μM). Expression of Akt was determined via western blot analysis and gels were loaded with equal amount of protein (120 μg). C: HC11 cells were infected with either a DN-Akt1 or control (LacZ) adenovirus. RNA was harvested 48 hours post-induction for analysis of β-casein RNA expression by northern blot. Expression of Akt was determined via western blot analysis and gels were loaded with equal amount of protein (120 μg).

Mentions: The activation of Akt is a major outcome of PI-3-kinase stimulation. Hence, the role of Akt in regulating HC11 lactogenic differentiation was examined. Transient transfection of a plasmid encoding a HA-tagged conditionally active-Akt-1 (CA-Akt1) gene was used to assess the ability of the activated Akt pathway to block lactogenic differentiation via inhibition of β-casein promotor luciferase activity [46]. HC11-luci cells were transiently transfected with either a plasmid encoding a HA-tagged conditionally active-Akt1 or a control vector. Western blotting of transfected cell lysates revealed that the HA-tagged conditionally active-Akt1 was expressed at levels equal to the endogenous Akt protein (figure 2A). The cells were induced to differentiate with DIP in the presence of 4-hydroxy-tamoxifen to activate the HA-tagged conditionally active-Akt1, and luciferase activity was determined 48 hours after induction. Expression of the conditionally active-Akt1 significantly decreased luciferase activity compared to the control vector and the addition of tamoxifen slightly reduced the luciferase activity in CA-Akt1 transfected cells (figure 2A). This indicated that the CA-Akt1 was not completely responsive to 4-hydroxy-tamoxifen under these conditions but that there was sufficient activity from the protein to activate PI-3-kinase signaling above that in control cells. The results in figure 4D confirm elevated activation of the pathway.


EGF-induced activation of Akt results in mTOR-dependent p70S6 kinase phosphorylation and inhibition of HC11 cell lactogenic differentiation.

Galbaugh T, Cerrito MG, Jose CC, Cutler ML - BMC Cell Biol. (2006)

The effect of conditionally active-Akt1 and dominant negative-Akt1 on HC11 differentiation. A. The HC11-luci cells transiently transfected with either a conditionally active-Akt-1 (CA-Akt) or a control vector (pCDNA3.1). At 24 hours the cells were incubated in DIP-induction media with or without tamoxifen (1 μM). Luciferase activity was determined 48 hours post-induction and was normalized to protein concentration. Expression of Akt and HA-CA-Akt was determined via western blot. Lanes were loaded with equal amounts of protein (117.5 μg). B. The effect of dominant negative-Akt1 (DN-Akt) adenoviral infection on EGF disruption of differentiation. HC11-luci cells were infected with either a DN-Akt1 or control (LacZ) adenovirus. Cells were changed to DIP-induction media the next day and lysates were harvested 48 hours post-induction for β-casein promotor luciferase activity. The results were compared to cultures exposed to DIP plus LY294002 (5 μM). Expression of Akt was determined via western blot analysis and gels were loaded with equal amount of protein (120 μg). C: HC11 cells were infected with either a DN-Akt1 or control (LacZ) adenovirus. RNA was harvested 48 hours post-induction for analysis of β-casein RNA expression by northern blot. Expression of Akt was determined via western blot analysis and gels were loaded with equal amount of protein (120 μg).
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Related In: Results  -  Collection

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Figure 2: The effect of conditionally active-Akt1 and dominant negative-Akt1 on HC11 differentiation. A. The HC11-luci cells transiently transfected with either a conditionally active-Akt-1 (CA-Akt) or a control vector (pCDNA3.1). At 24 hours the cells were incubated in DIP-induction media with or without tamoxifen (1 μM). Luciferase activity was determined 48 hours post-induction and was normalized to protein concentration. Expression of Akt and HA-CA-Akt was determined via western blot. Lanes were loaded with equal amounts of protein (117.5 μg). B. The effect of dominant negative-Akt1 (DN-Akt) adenoviral infection on EGF disruption of differentiation. HC11-luci cells were infected with either a DN-Akt1 or control (LacZ) adenovirus. Cells were changed to DIP-induction media the next day and lysates were harvested 48 hours post-induction for β-casein promotor luciferase activity. The results were compared to cultures exposed to DIP plus LY294002 (5 μM). Expression of Akt was determined via western blot analysis and gels were loaded with equal amount of protein (120 μg). C: HC11 cells were infected with either a DN-Akt1 or control (LacZ) adenovirus. RNA was harvested 48 hours post-induction for analysis of β-casein RNA expression by northern blot. Expression of Akt was determined via western blot analysis and gels were loaded with equal amount of protein (120 μg).
Mentions: The activation of Akt is a major outcome of PI-3-kinase stimulation. Hence, the role of Akt in regulating HC11 lactogenic differentiation was examined. Transient transfection of a plasmid encoding a HA-tagged conditionally active-Akt-1 (CA-Akt1) gene was used to assess the ability of the activated Akt pathway to block lactogenic differentiation via inhibition of β-casein promotor luciferase activity [46]. HC11-luci cells were transiently transfected with either a plasmid encoding a HA-tagged conditionally active-Akt1 or a control vector. Western blotting of transfected cell lysates revealed that the HA-tagged conditionally active-Akt1 was expressed at levels equal to the endogenous Akt protein (figure 2A). The cells were induced to differentiate with DIP in the presence of 4-hydroxy-tamoxifen to activate the HA-tagged conditionally active-Akt1, and luciferase activity was determined 48 hours after induction. Expression of the conditionally active-Akt1 significantly decreased luciferase activity compared to the control vector and the addition of tamoxifen slightly reduced the luciferase activity in CA-Akt1 transfected cells (figure 2A). This indicated that the CA-Akt1 was not completely responsive to 4-hydroxy-tamoxifen under these conditions but that there was sufficient activity from the protein to activate PI-3-kinase signaling above that in control cells. The results in figure 4D confirm elevated activation of the pathway.

Bottom Line: The activation of p70S6K by insulin or EGF resulted in the phosphorylation of ribosomal protein S6 (RPS6), elongation initiation factor 4E (elF4E) and 4E binding protein1 (4E-BP1).PI-3-kinase signaling contributes to the EGF block of lactogenic differentiation via Akt and p70S6K.The EGF-induced activation of PI-3-kinase-Akt-mTOR regulates phosphorylation of molecules including ribosomal protein S6, eIF4E and 4E-BP1 that influence translational control in HC11 cells undergoing lactogenic differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, United States Military Cancer Institute, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA. t-galbaugh@northwestern.edu

ABSTRACT

Background: HC11 mouse mammary epithelial cells differentiate in response to lactogenic hormone resulting in expression of milk proteins including beta-casein. Previous studies have shown that epidermal growth factor (EGF) blocks differentiation not only through activation of the Ras/Mek/Erk pathway but also implicated phosphatidylinositol-3-kinase (PI-3-kinase) signaling. The current study analyzes the mechanism of the PI-3-kinase pathway in an EGF-induced block of HC11 lactogenic differentiation.

Results: HC11 and HC11-luci cells, which contain luciferase gene under the control of a beta-casein promotor, were treated with specific chemical inhibitors of signal transduction pathways or transiently infected/transfected with vectors encoding dominant negative-Akt (DN-Akt) or conditionally active-Akt (CA-Akt). The expression of CA-Akt inhibited lactogenic differentiation of HC11 cells, and the infection with DN-Akt adenovirus enhanced beta-casein transcription and rescued beta-casein promotor-regulated luciferase activity in the presence of EGF. Treatment of cells with Rapamycin, an inhibitor of mTOR, blocked the effects of EGF on beta-casein promotor driven luciferase activity as effectively as PI-3-kinase inhibitors. While expression of CA-Akt caused a constitutive activation of p70S6 kinase (p70S6K) in HC11 cells, the inhibition of either PI-3-kinase or mTOR abolished the activation of p70S6K by EGF. The activation of p70S6K by insulin or EGF resulted in the phosphorylation of ribosomal protein S6 (RPS6), elongation initiation factor 4E (elF4E) and 4E binding protein1 (4E-BP1). But lower levels of PI-3-K and mTOR inhibitors were required to block insulin-induced phosphorylation of RPS6 than EGF-induced phosphorylation, and insulin-induced phosphorylation of elF4E and 4E-BP1 was not completely mTOR dependent suggesting some diversity of signaling for EGF and insulin. In HC11 cells undergoing lactogenic differentiation the phosphorylation of p70S6K completely diminished by 12 hours, and this was partly attributable to dexamethasone, a component of lactogenic hormone mix. However, p70S6K phosphorylation persisted in the presence of lactogenic hormone and EGF, but the activation could be blocked by a PI-3-kinase inhibitor.

Conclusion: PI-3-kinase signaling contributes to the EGF block of lactogenic differentiation via Akt and p70S6K. The EGF-induced activation of PI-3-kinase-Akt-mTOR regulates phosphorylation of molecules including ribosomal protein S6, eIF4E and 4E-BP1 that influence translational control in HC11 cells undergoing lactogenic differentiation.

Show MeSH
Related in: MedlinePlus