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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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Inhibition of ERK signaling inhibits migration of Akt1 down-regulated cells. (A) Migration of IGF-IR cells expressing Akt1 shRNA or empty vector (V) control treated with DMSO, 2–10 μM UO126, or 10 μM SB 202190 was assessed using transwell migration assay. Cells were starved in the absence of EGF. Migration was assessed after 16–20 h in media containing 2% horse serum, 100 ng/ml IGF-I, and DMSO or inhibitor. The histogram displays the mean percentage migration relative to Akt1 down-regulated IGF-IR cells treated with DMSO within the same experiment. The mean values were derived from three independent experiments. Error bars represent means ± SD. (B) Akt1 down-regulated IGF-IR cells were grown in 2% serum, 100 ng/ml IGF-I, and DMSO, 2–10 μM UO126, or SB 202190 for 72 h, with the inhibitor replaced after 48 h. Lysates were immunoblotted as indicated. (C) IGF-IR cells expressing Akt1 or empty vector shRNA were cultured in 2% serum, IGF-I, and DMSO or 2–10 μM UO126 for 72 h, lysed in NP-40 lysis buffer, and immunoblotted with antibodies against E-cadherin. (D) IGF-IR cells expressing Akt1 or empty vector shRNA and cultured in 2% serum, IGF-I, and DMSO or 2–10 μM UO126 for 72 h were lysed in RIPA lysis buffer and immunoblotted with the indicated antibodies.
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fig7: Inhibition of ERK signaling inhibits migration of Akt1 down-regulated cells. (A) Migration of IGF-IR cells expressing Akt1 shRNA or empty vector (V) control treated with DMSO, 2–10 μM UO126, or 10 μM SB 202190 was assessed using transwell migration assay. Cells were starved in the absence of EGF. Migration was assessed after 16–20 h in media containing 2% horse serum, 100 ng/ml IGF-I, and DMSO or inhibitor. The histogram displays the mean percentage migration relative to Akt1 down-regulated IGF-IR cells treated with DMSO within the same experiment. The mean values were derived from three independent experiments. Error bars represent means ± SD. (B) Akt1 down-regulated IGF-IR cells were grown in 2% serum, 100 ng/ml IGF-I, and DMSO, 2–10 μM UO126, or SB 202190 for 72 h, with the inhibitor replaced after 48 h. Lysates were immunoblotted as indicated. (C) IGF-IR cells expressing Akt1 or empty vector shRNA were cultured in 2% serum, IGF-I, and DMSO or 2–10 μM UO126 for 72 h, lysed in NP-40 lysis buffer, and immunoblotted with antibodies against E-cadherin. (D) IGF-IR cells expressing Akt1 or empty vector shRNA and cultured in 2% serum, IGF-I, and DMSO or 2–10 μM UO126 for 72 h were lysed in RIPA lysis buffer and immunoblotted with the indicated antibodies.

Mentions: To determine whether enhanced ERK activation is required for the phenotypes induced by Akt1 down-regulation, we examined the effects of inhibiting MEK-induced ERK activation using UO126, a specific MEK inhibitor. Treatment with UO126 significantly inhibited migration of Akt1 down-regulated cells, whereas treatment with a p38 inhibitor (SB 202190) or DMSO vehicle control had no effect (Fig. 7 A). Furthermore, this inhibition was observed at low concentrations of UO126 (2 μM), which reduced IGF-I–stimulated ERK activation in Akt1 down-regulated IGF-IR cells to the levels observed in IGF-IR control cells (Fig. 7 B); thus, these effects are detectable at levels of ERK inhibition that would not be expected to affect basal cell functions.


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)

Inhibition of ERK signaling inhibits migration of Akt1 down-regulated cells. (A) Migration of IGF-IR cells expressing Akt1 shRNA or empty vector (V) control treated with DMSO, 2–10 μM UO126, or 10 μM SB 202190 was assessed using transwell migration assay. Cells were starved in the absence of EGF. Migration was assessed after 16–20 h in media containing 2% horse serum, 100 ng/ml IGF-I, and DMSO or inhibitor. The histogram displays the mean percentage migration relative to Akt1 down-regulated IGF-IR cells treated with DMSO within the same experiment. The mean values were derived from three independent experiments. Error bars represent means ± SD. (B) Akt1 down-regulated IGF-IR cells were grown in 2% serum, 100 ng/ml IGF-I, and DMSO, 2–10 μM UO126, or SB 202190 for 72 h, with the inhibitor replaced after 48 h. Lysates were immunoblotted as indicated. (C) IGF-IR cells expressing Akt1 or empty vector shRNA were cultured in 2% serum, IGF-I, and DMSO or 2–10 μM UO126 for 72 h, lysed in NP-40 lysis buffer, and immunoblotted with antibodies against E-cadherin. (D) IGF-IR cells expressing Akt1 or empty vector shRNA and cultured in 2% serum, IGF-I, and DMSO or 2–10 μM UO126 for 72 h were lysed in RIPA lysis buffer and immunoblotted with the indicated antibodies.
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fig7: Inhibition of ERK signaling inhibits migration of Akt1 down-regulated cells. (A) Migration of IGF-IR cells expressing Akt1 shRNA or empty vector (V) control treated with DMSO, 2–10 μM UO126, or 10 μM SB 202190 was assessed using transwell migration assay. Cells were starved in the absence of EGF. Migration was assessed after 16–20 h in media containing 2% horse serum, 100 ng/ml IGF-I, and DMSO or inhibitor. The histogram displays the mean percentage migration relative to Akt1 down-regulated IGF-IR cells treated with DMSO within the same experiment. The mean values were derived from three independent experiments. Error bars represent means ± SD. (B) Akt1 down-regulated IGF-IR cells were grown in 2% serum, 100 ng/ml IGF-I, and DMSO, 2–10 μM UO126, or SB 202190 for 72 h, with the inhibitor replaced after 48 h. Lysates were immunoblotted as indicated. (C) IGF-IR cells expressing Akt1 or empty vector shRNA were cultured in 2% serum, IGF-I, and DMSO or 2–10 μM UO126 for 72 h, lysed in NP-40 lysis buffer, and immunoblotted with antibodies against E-cadherin. (D) IGF-IR cells expressing Akt1 or empty vector shRNA and cultured in 2% serum, IGF-I, and DMSO or 2–10 μM UO126 for 72 h were lysed in RIPA lysis buffer and immunoblotted with the indicated antibodies.
Mentions: To determine whether enhanced ERK activation is required for the phenotypes induced by Akt1 down-regulation, we examined the effects of inhibiting MEK-induced ERK activation using UO126, a specific MEK inhibitor. Treatment with UO126 significantly inhibited migration of Akt1 down-regulated cells, whereas treatment with a p38 inhibitor (SB 202190) or DMSO vehicle control had no effect (Fig. 7 A). Furthermore, this inhibition was observed at low concentrations of UO126 (2 μM), which reduced IGF-I–stimulated ERK activation in Akt1 down-regulated IGF-IR cells to the levels observed in IGF-IR control cells (Fig. 7 B); thus, these effects are detectable at levels of ERK inhibition that would not be expected to affect basal cell functions.

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