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Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transition.

Irie HY, Pearline RV, Grueneberg D, Hsia M, Ravichandran P, Kothari N, Natesan S, Brugge JS - J. Cell Biol. (2005)

Bottom Line: In contrast, Akt1 down-regulation in IGF-IR-stimulated cells promoted dramatic neomorphic effects characteristic of an epithelial-mesenchymal transition (EMT) and enhanced cell migration induced by IGF-I or EGF stimulation.The phenotypic effects of Akt1 down-regulation were accompanied by enhanced extracellular signal-related kinase (ERK) activation, which contributed to the induction of migration and EMT.These results highlight the distinct functions of Akt isoforms in regulating growth factor-stimulated EMT and cell migration, as well as the importance of Akt1 in cross-regulating the ERK signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The Akt family of kinases are activated by growth factors and regulate pleiotropic cellular activities. In this study, we provide evidence for isoform-specific positive and negative roles for Akt1 and -2 in regulating growth factor-stimulated phenotypes in breast epithelial cells. Insulin-like growth factor-I receptor (IGF-IR) hyperstimulation induced hyperproliferation and antiapoptotic activities that were reversed by Akt2 down-regulation. In contrast, Akt1 down-regulation in IGF-IR-stimulated cells promoted dramatic neomorphic effects characteristic of an epithelial-mesenchymal transition (EMT) and enhanced cell migration induced by IGF-I or EGF stimulation. The phenotypic effects of Akt1 down-regulation were accompanied by enhanced extracellular signal-related kinase (ERK) activation, which contributed to the induction of migration and EMT. Interestingly, down-regulation of Akt2 suppressed the EMT-like morphological conversion induced by Akt1 down-regulation in IGF-IR-overexpressing cells and inhibited migration in EGF-stimulated cells. These results highlight the distinct functions of Akt isoforms in regulating growth factor-stimulated EMT and cell migration, as well as the importance of Akt1 in cross-regulating the ERK signaling pathway.

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Isoform-specific down-regulation of Akt results in distinct phenotypes. (A) IGF-IR cells were superinfected with shRNA vectors targeting Akt1 and/or -2. Empty vectors (V) were used as controls. Immunoblotting with antibody against actin was used to confirm equal loading. Cells in monolayer cultures were lysed and immunoblotted with Akt isoform-specific antibodies. (B) IGF-IR cells superinfected with empty vector, Akt1 shRNA, Akt2 shRNA, or both were grown in triplicate monolayer cultures for the indicated days in media containing EGF and IGF-I (100 ng/ml), with initial seeding of 2.5 × 104 cells. Cells were trypsinized, stained with Trypan blue, and counted. A representative experiment of three independent experiments is shown. Error bars represent means ± SD. (C) IGF-IR cells in which either Akt1 and/or -2 were down-regulated were cultured in monolayer (top) or 3D Matrigel/collagen (50:50) cultures for 8 d (middle and bottom). Monolayer cultures were grown in the presence of EGF and IGF-I (100 ng/ml). 3D cultures were grown in the presence of 5 ng/ml EGF and 100 ng/ml IGF-I. Bars: (top and middle) 50 μM; (bottom) 100 μM.
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fig2: Isoform-specific down-regulation of Akt results in distinct phenotypes. (A) IGF-IR cells were superinfected with shRNA vectors targeting Akt1 and/or -2. Empty vectors (V) were used as controls. Immunoblotting with antibody against actin was used to confirm equal loading. Cells in monolayer cultures were lysed and immunoblotted with Akt isoform-specific antibodies. (B) IGF-IR cells superinfected with empty vector, Akt1 shRNA, Akt2 shRNA, or both were grown in triplicate monolayer cultures for the indicated days in media containing EGF and IGF-I (100 ng/ml), with initial seeding of 2.5 × 104 cells. Cells were trypsinized, stained with Trypan blue, and counted. A representative experiment of three independent experiments is shown. Error bars represent means ± SD. (C) IGF-IR cells in which either Akt1 and/or -2 were down-regulated were cultured in monolayer (top) or 3D Matrigel/collagen (50:50) cultures for 8 d (middle and bottom). Monolayer cultures were grown in the presence of EGF and IGF-I (100 ng/ml). 3D cultures were grown in the presence of 5 ng/ml EGF and 100 ng/ml IGF-I. Bars: (top and middle) 50 μM; (bottom) 100 μM.

Mentions: To investigate whether specific Akt isoforms are critical for the phenotypes induced by IGF-IR in the 3D culture model, we used RNA interference to specifically down-regulate the expression of Akt isoforms. Quantitative analyses using purified Akt1, -2, and -3 as protein standards revealed that Akt1 is present in excess of Akt2 (approximately threefold) and -3 (slightly less than twofold; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200505087/DC1). Retroviral or lentiviral vectors encoding short hairpin RNA (shRNA) sequences were used to specifically down-regulate the expression of Akt1 and/or -2 in cells overexpressing wild-type IGF-IR (Fig. 2 A). Although several shRNA vectors targeting Akt3 were evaluated, none consistently down-regulated Akt3 expression. The down-regulation of Akt1 or -2 using shRNA vectors was at least 75% (Fig. 2 A and Fig. S1) and confirmed to be stable for at least 2 wks (not depicted).


Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transition.

Irie HY, Pearline RV, Grueneberg D, Hsia M, Ravichandran P, Kothari N, Natesan S, Brugge JS - J. Cell Biol. (2005)

Isoform-specific down-regulation of Akt results in distinct phenotypes. (A) IGF-IR cells were superinfected with shRNA vectors targeting Akt1 and/or -2. Empty vectors (V) were used as controls. Immunoblotting with antibody against actin was used to confirm equal loading. Cells in monolayer cultures were lysed and immunoblotted with Akt isoform-specific antibodies. (B) IGF-IR cells superinfected with empty vector, Akt1 shRNA, Akt2 shRNA, or both were grown in triplicate monolayer cultures for the indicated days in media containing EGF and IGF-I (100 ng/ml), with initial seeding of 2.5 × 104 cells. Cells were trypsinized, stained with Trypan blue, and counted. A representative experiment of three independent experiments is shown. Error bars represent means ± SD. (C) IGF-IR cells in which either Akt1 and/or -2 were down-regulated were cultured in monolayer (top) or 3D Matrigel/collagen (50:50) cultures for 8 d (middle and bottom). Monolayer cultures were grown in the presence of EGF and IGF-I (100 ng/ml). 3D cultures were grown in the presence of 5 ng/ml EGF and 100 ng/ml IGF-I. Bars: (top and middle) 50 μM; (bottom) 100 μM.
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Related In: Results  -  Collection

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fig2: Isoform-specific down-regulation of Akt results in distinct phenotypes. (A) IGF-IR cells were superinfected with shRNA vectors targeting Akt1 and/or -2. Empty vectors (V) were used as controls. Immunoblotting with antibody against actin was used to confirm equal loading. Cells in monolayer cultures were lysed and immunoblotted with Akt isoform-specific antibodies. (B) IGF-IR cells superinfected with empty vector, Akt1 shRNA, Akt2 shRNA, or both were grown in triplicate monolayer cultures for the indicated days in media containing EGF and IGF-I (100 ng/ml), with initial seeding of 2.5 × 104 cells. Cells were trypsinized, stained with Trypan blue, and counted. A representative experiment of three independent experiments is shown. Error bars represent means ± SD. (C) IGF-IR cells in which either Akt1 and/or -2 were down-regulated were cultured in monolayer (top) or 3D Matrigel/collagen (50:50) cultures for 8 d (middle and bottom). Monolayer cultures were grown in the presence of EGF and IGF-I (100 ng/ml). 3D cultures were grown in the presence of 5 ng/ml EGF and 100 ng/ml IGF-I. Bars: (top and middle) 50 μM; (bottom) 100 μM.
Mentions: To investigate whether specific Akt isoforms are critical for the phenotypes induced by IGF-IR in the 3D culture model, we used RNA interference to specifically down-regulate the expression of Akt isoforms. Quantitative analyses using purified Akt1, -2, and -3 as protein standards revealed that Akt1 is present in excess of Akt2 (approximately threefold) and -3 (slightly less than twofold; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200505087/DC1). Retroviral or lentiviral vectors encoding short hairpin RNA (shRNA) sequences were used to specifically down-regulate the expression of Akt1 and/or -2 in cells overexpressing wild-type IGF-IR (Fig. 2 A). Although several shRNA vectors targeting Akt3 were evaluated, none consistently down-regulated Akt3 expression. The down-regulation of Akt1 or -2 using shRNA vectors was at least 75% (Fig. 2 A and Fig. S1) and confirmed to be stable for at least 2 wks (not depicted).

Bottom Line: In contrast, Akt1 down-regulation in IGF-IR-stimulated cells promoted dramatic neomorphic effects characteristic of an epithelial-mesenchymal transition (EMT) and enhanced cell migration induced by IGF-I or EGF stimulation.The phenotypic effects of Akt1 down-regulation were accompanied by enhanced extracellular signal-related kinase (ERK) activation, which contributed to the induction of migration and EMT.These results highlight the distinct functions of Akt isoforms in regulating growth factor-stimulated EMT and cell migration, as well as the importance of Akt1 in cross-regulating the ERK signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The Akt family of kinases are activated by growth factors and regulate pleiotropic cellular activities. In this study, we provide evidence for isoform-specific positive and negative roles for Akt1 and -2 in regulating growth factor-stimulated phenotypes in breast epithelial cells. Insulin-like growth factor-I receptor (IGF-IR) hyperstimulation induced hyperproliferation and antiapoptotic activities that were reversed by Akt2 down-regulation. In contrast, Akt1 down-regulation in IGF-IR-stimulated cells promoted dramatic neomorphic effects characteristic of an epithelial-mesenchymal transition (EMT) and enhanced cell migration induced by IGF-I or EGF stimulation. The phenotypic effects of Akt1 down-regulation were accompanied by enhanced extracellular signal-related kinase (ERK) activation, which contributed to the induction of migration and EMT. Interestingly, down-regulation of Akt2 suppressed the EMT-like morphological conversion induced by Akt1 down-regulation in IGF-IR-overexpressing cells and inhibited migration in EGF-stimulated cells. These results highlight the distinct functions of Akt isoforms in regulating growth factor-stimulated EMT and cell migration, as well as the importance of Akt1 in cross-regulating the ERK signaling pathway.

Show MeSH
Related in: MedlinePlus