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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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Down-regulation of Akt1 in IGF-IR cells induces migration and EMT. (A, top) The motility of IGF-IR cells with isoform-specific down-regulation of Akt was assessed using transwell assays. Cells were starved overnight in 2% serum in the absence of EGF and IGF. Migration was assessed after 16–20 h in the presence or absence of 100 ng/ml IGF-I. The histogram displays the mean number of migrated cells obtained by counting 10 separate fields in three independent experiments. (bottom) IGF-IR cells with down-regulation of both Akt1 and -2 were starved and migration in the presence or absence of IGF-I was assessed. Shown is a representative experiment displaying the mean number of migrated cells in five separate fields. Error bars represent means ± SD. (B) IGF-IR cells infected with empty vector control (V) or shRNA vectors targeting Akt1 or -2 were grown in 2% horse serum and 100 ng/ml IGF-I and lysed in NP-40 (for E- and N-cadherin) or RIPA buffer (Vimentin). Lysates were immunoblotted with the indicated antibodies.
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fig4: Down-regulation of Akt1 in IGF-IR cells induces migration and EMT. (A, top) The motility of IGF-IR cells with isoform-specific down-regulation of Akt was assessed using transwell assays. Cells were starved overnight in 2% serum in the absence of EGF and IGF. Migration was assessed after 16–20 h in the presence or absence of 100 ng/ml IGF-I. The histogram displays the mean number of migrated cells obtained by counting 10 separate fields in three independent experiments. (bottom) IGF-IR cells with down-regulation of both Akt1 and -2 were starved and migration in the presence or absence of IGF-I was assessed. Shown is a representative experiment displaying the mean number of migrated cells in five separate fields. Error bars represent means ± SD. (B) IGF-IR cells infected with empty vector control (V) or shRNA vectors targeting Akt1 or -2 were grown in 2% horse serum and 100 ng/ml IGF-I and lysed in NP-40 (for E- and N-cadherin) or RIPA buffer (Vimentin). Lysates were immunoblotted with the indicated antibodies.

Mentions: To establish whether Akt1 down-regulation also affects the migratory behavior of IGF-IR cells, as well as their invasive activity in basement membrane cultures, we examined cell motility in transwell assays. Ligand stimulation of control or IGF-IR cells did not stimulate migration in transwell assays. (Fig. 4 A, top). However, down-regulation of Akt1 with either shRNA sequence (sequence A or B) caused a dramatic enhancement of cell migration relative to IGF-IR cells superinfected with control empty vectors. In contrast, down-regulation of Akt2 did not enhance migration of IGF-IR cells. The increase in migration observed with Akt1 down-regulation occurred under basal conditions, but was significantly enhanced by IGF-I stimulation. The enhanced basal and IGF-I–stimulated migration induced by Akt1 down-regulation was impaired with concomitant Akt2 down-regulation (Fig. 4 A, bottom), suggesting that Akt2 is required for this effect of Akt1 down-regulation.


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)

Down-regulation of Akt1 in IGF-IR cells induces migration and EMT. (A, top) The motility of IGF-IR cells with isoform-specific down-regulation of Akt was assessed using transwell assays. Cells were starved overnight in 2% serum in the absence of EGF and IGF. Migration was assessed after 16–20 h in the presence or absence of 100 ng/ml IGF-I. The histogram displays the mean number of migrated cells obtained by counting 10 separate fields in three independent experiments. (bottom) IGF-IR cells with down-regulation of both Akt1 and -2 were starved and migration in the presence or absence of IGF-I was assessed. Shown is a representative experiment displaying the mean number of migrated cells in five separate fields. Error bars represent means ± SD. (B) IGF-IR cells infected with empty vector control (V) or shRNA vectors targeting Akt1 or -2 were grown in 2% horse serum and 100 ng/ml IGF-I and lysed in NP-40 (for E- and N-cadherin) or RIPA buffer (Vimentin). Lysates were immunoblotted with the indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171329&req=5

fig4: Down-regulation of Akt1 in IGF-IR cells induces migration and EMT. (A, top) The motility of IGF-IR cells with isoform-specific down-regulation of Akt was assessed using transwell assays. Cells were starved overnight in 2% serum in the absence of EGF and IGF. Migration was assessed after 16–20 h in the presence or absence of 100 ng/ml IGF-I. The histogram displays the mean number of migrated cells obtained by counting 10 separate fields in three independent experiments. (bottom) IGF-IR cells with down-regulation of both Akt1 and -2 were starved and migration in the presence or absence of IGF-I was assessed. Shown is a representative experiment displaying the mean number of migrated cells in five separate fields. Error bars represent means ± SD. (B) IGF-IR cells infected with empty vector control (V) or shRNA vectors targeting Akt1 or -2 were grown in 2% horse serum and 100 ng/ml IGF-I and lysed in NP-40 (for E- and N-cadherin) or RIPA buffer (Vimentin). Lysates were immunoblotted with the indicated antibodies.
Mentions: To establish whether Akt1 down-regulation also affects the migratory behavior of IGF-IR cells, as well as their invasive activity in basement membrane cultures, we examined cell motility in transwell assays. Ligand stimulation of control or IGF-IR cells did not stimulate migration in transwell assays. (Fig. 4 A, top). However, down-regulation of Akt1 with either shRNA sequence (sequence A or B) caused a dramatic enhancement of cell migration relative to IGF-IR cells superinfected with control empty vectors. In contrast, down-regulation of Akt2 did not enhance migration of IGF-IR cells. The increase in migration observed with Akt1 down-regulation occurred under basal conditions, but was significantly enhanced by IGF-I stimulation. The enhanced basal and IGF-I–stimulated migration induced by Akt1 down-regulation was impaired with concomitant Akt2 down-regulation (Fig. 4 A, bottom), suggesting that Akt2 is required for this effect of Akt1 down-regulation.

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