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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.

Show MeSH
Akt2-specific down-regulation inhibits IGF-IR–induced changes in 3D cultures. IGF-IR cells expressing Akt2 shRNA vector were cultured in 3D Matrigel cultures for 8 or 16 d in the presence of EGF and IGF-I (100 ng/ml). Structures were stained with antibodies to cleaved caspase-3 or Ki-67 (green), α6 integrin (red), and TOPRO (blue). Equatorial confocal images are shown. Bars, 50 μM.
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fig3: Akt2-specific down-regulation inhibits IGF-IR–induced changes in 3D cultures. IGF-IR cells expressing Akt2 shRNA vector were cultured in 3D Matrigel cultures for 8 or 16 d in the presence of EGF and IGF-I (100 ng/ml). Structures were stained with antibodies to cleaved caspase-3 or Ki-67 (green), α6 integrin (red), and TOPRO (blue). Equatorial confocal images are shown. Bars, 50 μM.

Mentions: In 3D Matrigel/collagen (50:50) cultures (Fig. 2 C, middle and bottom), Akt2 down-regulation suppressed the IGF-IR–induced hyperproliferative phenotype, resulting in structures that resembled parental MCF-10A acini, although slightly larger (Fig. 2 C, compare h with g and f). We also examined the effects of Akt2 down-regulation on IGF-IR–induced hyperproliferation, by examining expression of Ki67, a marker of cycling cells, and on luminal apoptosis by examining caspase-3 cleavage. IGF-IR structures exhibited significant Ki67 staining at day 16, unlike parental MCF-10A cultures, which have arrested proliferation by this time period (Debnath et al., 2002). In contrast, Akt2–down-regulated IGF-IR structures underwent proliferative arrest similar to that of parental MCF-10A acini, as demonstrated by the absence of Ki67 staining at day 16. (Fig. 3, bottom). IGF-IR 3D structures cultured in the presence of IGF-I also exhibit filled lumen because of their failure to undergo cavitation via apoptosis of centrally localized cells, a key feature of morphogenesis of parental MCF-10A acini (Debnath et al., 2002). Suppression of Akt2 expression restored lumen formation and luminal apoptosis, with intense activated caspase-3 staining in the presumptive luminal space (Fig. 3).


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)

Akt2-specific down-regulation inhibits IGF-IR–induced changes in 3D cultures. IGF-IR cells expressing Akt2 shRNA vector were cultured in 3D Matrigel cultures for 8 or 16 d in the presence of EGF and IGF-I (100 ng/ml). Structures were stained with antibodies to cleaved caspase-3 or Ki-67 (green), α6 integrin (red), and TOPRO (blue). Equatorial confocal images are shown. Bars, 50 μM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171329&req=5

fig3: Akt2-specific down-regulation inhibits IGF-IR–induced changes in 3D cultures. IGF-IR cells expressing Akt2 shRNA vector were cultured in 3D Matrigel cultures for 8 or 16 d in the presence of EGF and IGF-I (100 ng/ml). Structures were stained with antibodies to cleaved caspase-3 or Ki-67 (green), α6 integrin (red), and TOPRO (blue). Equatorial confocal images are shown. Bars, 50 μM.
Mentions: In 3D Matrigel/collagen (50:50) cultures (Fig. 2 C, middle and bottom), Akt2 down-regulation suppressed the IGF-IR–induced hyperproliferative phenotype, resulting in structures that resembled parental MCF-10A acini, although slightly larger (Fig. 2 C, compare h with g and f). We also examined the effects of Akt2 down-regulation on IGF-IR–induced hyperproliferation, by examining expression of Ki67, a marker of cycling cells, and on luminal apoptosis by examining caspase-3 cleavage. IGF-IR structures exhibited significant Ki67 staining at day 16, unlike parental MCF-10A cultures, which have arrested proliferation by this time period (Debnath et al., 2002). In contrast, Akt2–down-regulated IGF-IR structures underwent proliferative arrest similar to that of parental MCF-10A acini, as demonstrated by the absence of Ki67 staining at day 16. (Fig. 3, bottom). IGF-IR 3D structures cultured in the presence of IGF-I also exhibit filled lumen because of their failure to undergo cavitation via apoptosis of centrally localized cells, a key feature of morphogenesis of parental MCF-10A acini (Debnath et al., 2002). Suppression of Akt2 expression restored lumen formation and luminal apoptosis, with intense activated caspase-3 staining in the presumptive luminal space (Fig. 3).

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